Pesticidal proteins

ABSTRACT

The subject invention concerns new classes of pesticidally active proteins and the polynucleotide sequences that encode these proteins. In preferred embodiments, these pesticidal protein have molecular weights of approximately 40-50 kDa and of approximately 10-15 kDa.

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application is a divisional of U.S. Ser. No. 09/643,596,filed Aug. 22, 2000, which is a continuation-in-part of U.S. Ser. No.09/378,088, filed Aug. 20, 1999, now U.S. Pat. No. 6,372,480, which is acontinuation-in-part of Ser. No. 08/844,188, filed Apr. 18, 1997, nowU.S. Pat. No. 6,127,180, which is a continuation-in-part of Ser. No.08/633,993, filed Apr. 19, 1996, which issued as U.S. Pat. No. 6,083,499on Jul. 4, 2000.

BACKGROUND OF THE INVENTION

[0002] Coleopterans are a significant group of agricultural pests whichcause extensive damage to crops each year. Examples of coleopteran pestsinclude corn rootworm and alfalfa weevils.

[0003] The alfalfa weevil, Hypera postica, and the closely relatedEgyptian alfalfa weevil, Hypera brunneipennis, are the most importantinsect pests of alfalfa grown in the United States, with 2.9 millionacres infested in 1984. An annual sum of 20 million dollars is spent tocontrol these pests. The Egyptian alfalfa weevil is the predominantspecies in the southwestern U.S., where it undergoes aestivation (i.e.,hibernation) during the hot summer months. In all other respects, it isidentical to the alfalfa weevil, which predominates throughout the restof the U.S.

[0004] The larval stage is the most damaging in the weevil life cycle.By feeding at the alfalfa plant's growing tips, the larvae causeskeletonization of leaves, stunting, reduced plant growth, and,ultimately, reductions in yield. Severe infestations can ruin an entirecutting of hay. The adults, also foliar feeders, cause additional, butless significant, damage.

[0005] Approximately 10 million acres of U.S. corn are infested withcorn rootworm species complex each year. The corn rootworm speciescomplex includes the northern corn rootworm, Diabrotica barberi, thesouthern corn rootworm, D. undecimpunctata howardi, and the western cornrootworm, D. virgifera virgifera. The soil-dwelling larvae of theseDiabrotica species feed on the root of the corn plant, causing lodging.Lodging eventually reduces corn yield and often results in death of theplant. By feeding on cornsilks, the adult beetles reduce pollinationand, therefore, detrimentally affect the yield of corn per plant. Inaddition, adults and larvae of the genus Diabrotica attack cucurbitcrops (cucumbers, melons, squash, etc.) and many vegetable and fieldcrops in commercial production as well as those being grown in homegardens.

[0006] Control of corn rootworm has been partially addressed bycultivation methods, such as crop rotation and the application of highnitrogen levels to stimulate the growth of an adventitious root system.However, chemical insecticides are relied upon most heavily to guaranteethe desired level of control. Insecticides are either banded onto orincorporated into the soil. Problems associated with the use of somechemical insecticides are environmental contamination and thedevelopment of resistance among the treated insect populations.

[0007] The soil microbe Bacillus thuringiensis (B.t.) is aGram-positive, spore-forming bacterium characterized by parasporalprotein inclusions, which can appear microscopically as distinctivelyshaped crystals. Certain strains of B.t. produce proteins that are toxicto specific orders of pests. Certain B.t. toxin genes have been isolatedand sequenced, and recombinant DNA-based B.t. products have beenproduced and approved for use. In addition, with the use of geneticengineering techniques, new approaches for delivering these B.t.endotoxins to agricultural environments are under development, includingthe use of plants genetically engineered with endotoxin genes for insectresistance and the use of stabilized intact microbial cells as B.t.endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commerciallyvaluable.

[0008] Commercial use of B.t. pesticides was originally limited to anarrow range of lepidopteran (caterpillar) pests. Preparations of thespores and crystals of B. thuringiensis subsp. kurstaki have been usedfor many years as commercial insecticides for lepidopteran pests. Forexample, B. thuringiensis var. kurstaki HD-1 produces a crystalline8-endotoxin which is toxic to the larvae of a number of lepidopteraninsects.

[0009] In recent years, however, investigators have discovered B.t.pesticides with specificities for a much broader range of pests. Forexample, other species of B.t., namely israelensis and tenebrionis(a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially tocontrol insects of the orders Diptera and Coleoptera, respectively(Gaertner, F. H. [1989] “Cellular Delivery Systems for InsecticidalProteins: Living and Non-Living Microorganisms,” in Controlled Deliveryof Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, NewYork and London, 1990, pp. 245-255). See also Couch, T. L. (1980)“Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,”Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978)“Use of Entomogenous Bacteria in Agroecosystems,” Developments inIndustrial Microbiology 20:97-104. Krieg, A., A. M. Huger, G. A.Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describeBacillus thuringiensis var. tenebrionis, which is reportedly activeagainst two beetles in the order Coleoptera. These are the Coloradopotato beetle, Leptinotarsa decemlineata, and Agelastica alni.

[0010] Recently, new subspecies of B.t. have been identified, and genesresponsible for active δ-endotoxin proteins have been isolated (Höfte,H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Höfteand Whiteley classified B.t. crystal protein genes into four majorclasses. The classes were CryI (Lepidoptera-specific), CryII(Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), andCryIV (Diptera-specific). The discovery of strains specifically toxic toother pests has been reported. (Feitelson, J. S., J. Payne, L. Kim[1992] Bio/Technology 10:271-275).

[0011] The 1989 nomenclature and classification scheme of Hofte andWhiteley for crystal proteins was based on both the deduced amino acidsequence and the host range of the toxin. That system was adapted tocover fourteen different types of toxin genes which were divided intofive major classes. As more toxin genes were discovered, that systemstarted to become unworkable, as genes with similar sequences were foundto have significantly different insecticidal specificities. A revisednomenclature scheme has been proposed which is based solely on aminoacid identity (Crickmore et al. [1996] Society for InvertebratePathology, 29th Annual Meeting, 3rd International Colloquium on Bacillusthuringiensis, University of Cordoba, Cordoba, Spain, September 1-6,abstract). The mnemonic “cry” has been retained for all of the toxingenes except cytA and cytB, which remain a separate class. Romannumerals have been exchanged for Arabic numerals in the primary rank,and the parentheses in the tertiary rank have been removed. Currentboundaries represent approximately 95% (tertiary rank), 75% (secondaryrank), and 48% (primary rank) sequence identity. Many of the originalnames have been retained, with the noted exceptions, although a numberhave been reclassified. See also N. Crickmore, D. R. Zeigler, J.Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean(1998), “Revisions of the Nomenclature for the Bacillus thuringiensisPesticidal Crystal Proteins,” Microbiology and Molecular Biology ReviewsVol. 62:807-813; and Crickmore, Zeigler, Feitelson, Schnepf, Van Rie,Lereclus, Baum, and Dean, “Bacillus thuringiensis toxin nomenclature”(1999) http://www.biols.susx.ac.uk/Home/Neil_(—)Crickmore/Bt/index.html.That system uses the freely available software applications CLUSTAL Wand PHYLIP. The NEIGHBOR application within the PHYLIP package uses anarithmetic averages (UPGMA) algorithm.

[0012] The cloning and expression of a B.t. crystal protein gene inEscherichia coli has been described in the published literature(Schnepf, H. E., H. R. Whiteley [1981] Proc. Natl. Acad. Sci. USA78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 bothdisclose the expression of B.t. crystal protein in E. coli.

[0013] U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensisstrain tenebrionis (a.k.a. M-7, a.k.a. B.t. san diego), which can beused to control coleopteran pests in various environments. U.S. Pat. No.4,918,006 discloses B.t. toxins having activity against Dipterans. U.S.Pat. No. 4,849,217 discloses B.t. isolates which have activity againstthe alfalfa weevil. U.S. Pat. No. 5,208,077 discloses coleopteran-activeBacillus thuringiensis isolates. U.S. Pat. No. 5,632,987 discloses a 130kDa toxin from PS80JJ1 as having activity against corn rootworm. WO94/40162, which is related to the subject application, describes newclasses of proteins that are toxic to corn rootworm. U.S. Pat. No.5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t.which have activity against nematodes.

[0014] U.S. Pat. No. 6,083,499 and WO 97/40162 disclose “binary toxins.”The subject invention is distinct from mosquitocidal toxins produced byBacillus sphaericus. See EP 454 485; Davidson et al. (1990),“Interaction of the Bacillus sphaericus mosquito larvicidal proteins,”Can. J Microbiol. 36 (12):870-8; Baumann et al. (1988), “Sequenceanalysis of the mosquitocidal toxin genes encoding 51.4- and41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297,” J.Bacteriol. 170:2045-2050; Oei et al. (1992), “Binding of purifiedBacillus sphaericus binary toxin and its deletion derivatives to Culexquinquefasciatus gut: elucidation of functional binding domains,”Journal of General Microbiology 138 (7): 1515-26.

BRIEF SUMMARY OF THE INVENTION

[0015] The subject invention concerns novel materials and methods forcontrolling non-mammalian pests. In a preferred embodiment, the subjectinvention provides materials and methods for the control of coleopteranpests. In more preferred embodiments, the materials and methodsdescribed herein are used to control corn rootworm—most preferablyWestern corn rootworm. Lepidopteran pests (including the European cornborer and Helicoverpa zea) can also be controlled by the pesticidalproteins of the subject invention.

[0016] The subject invention advantageously provides polynucleotides andpesticidal proteins encoded by the polynucleotides. In preferredembodiments, a 40-50 kDa protein and a 10-15 kDa protein are usedtogether, with the proteins being pesticidal in combination. Thus, thetwo classes of proteins of the subject invention can be referred to as“binary toxins.” As used herein, the term “toxin” or “pesticidalprotein” includes either class of these proteins. The use of a 40-50 kDaprotein with a 10-15 kDa protein is preferred but not necessarilyrequired. One class of polynucleotide sequences as described hereinencodes proteins which have a full-length molecular weight ofapproximately 40-50 kDa. In a specific embodiment, these proteins have amolecular weight of about 43-47 kDa. A second class of polynucleotidesof the subject invention encodes pesticidal proteins of about 10-15 kDa.In a specific embodiment, these proteins have a molecular weight ofabout 13-14 kDa. It should be clear that each type of toxin/gene is anaspect of the subject invention. In a particularly preferred embodiment,a 40-50 kDa protein of the subject invention is used in combination witha 10-15 kDa protein. Thus, the proteins of the subject invention can beused to augment and/or facilitate the activity of other protein toxins.

[0017] The subject invention includes polynucleotides that encode the40-50 kDa or the 10-15 kDa toxins, polynucleotides that encode portionsor fragments of the full length toxins that retain pesticidal activity(preferably when used in combination), and polynucleotides that encodeboth types of toxins. Novel examples of fusion proteins (a 40-50 kDaprotein and a 10-15 kDa protein fused together) and polynucleotides thatencode them are also disclosed herein.

[0018] In some embodiments, B.t. toxins useful according to theinvention include toxins which can be obtained from the novel B.t.isolates disclosed herein. It should be clear that, where 40-50 kDa and10-15 kDa toxins, for example, are used together, one type of toxin canbe obtained from one isolate and the other type of toxin can be obtainedfrom another isolate.

[0019] The subject invention also includes the use of variants of theexemplified B.t. isolates and toxins which have substantially the samecoleopteran-active properties as the specifically exemplified B.t.isolates and toxins. Such variant isolates would include, for example,mutants. Procedures for making mutants are well known in themicrobiological art. Ultraviolet light and chemical mutagens such asnitrosoguanidine are used extensively toward this end.

[0020] In preferred embodiments, the subject invention concerns plantsand plant cells having at least one isolated polynucleotide of thesubject invention. Preferably, the transgenic plant cells expresspesticidal toxins in tissues consumed by the target pests.

[0021] Alternatively, the B.t. isolates of the subject invention, orrecombinant microbes expressing the toxins described herein, can be usedto control pests. In this regard, the invention includes the treatmentof substantially intact B.t. cells, and/or recombinant cells containingthe expressed toxins of the invention, treated to prolong the pesticidalactivity when the substantially intact cells are applied to theenvironment of a target pest. The treated cell acts as a protectivecoating for the pesticidal toxin.

[0022] The toxins of the subject invention are oral intoxicants thataffect an insect's midgut cells upon ingestion by the target insect.Thus, by consuming recombinant host cells, for example, that express thetoxins, the target insect thereby contacts the proteins of the subjectinvention, which are toxic to the pest. This results in control of thetarget pest.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows three exemplary 43-47 kDa pesticidal toxins as wellas a consensus sequence for these pesticidal toxins.

[0024]FIG. 2 shows the relationship of the 14 and 45 kDa sequences ofPS80JJ1 (SEQ ID NOS. 31 and 10).

[0025]FIG. 3 shows a comparison of LC₅₀ values from the mixing study ofExample 23.

[0026]FIG. 4 shows protein alignments of the 51 and 42 kDa Bacillussphaericus toxins and genes and the 45 kDa 149B1 toxin and gene.

[0027]FIG. 5 shows nucleotide sequence alignments of the 51 and 42 kDaBacillus sphaericus toxins and genes and the 45 kDa 149B1 toxin andgene.

BRIEF DESCRIPTION OF THE SEQUENCES

[0028] SEQ ID NO:1 is a 5-amino acid N-terminal sequence of theapproximately 45 kDa toxin of 80JJ1.

[0029] SEQ ID NO:2 is a 25-amino acid N-terminal sequence of theapproximately 45 kDa toxin of 80JJ1.

[0030] SEQ ID NO:3 is a 24-amino acid N-terminal sequence of theapproximately 14 kDa toxin of 80JJ1.

[0031] SEQ ID NO:4 is the N-terminal sequence of the approximately 47kDa toxin from 149B1.

[0032] SEQ ID NO:5 is a 50-amino acid N-terminal amino acid sequence forthe purified approximately 14 kDa protein from PS149B1.

[0033] SEQ ID NO:6 is the N-terminal sequence of the approximately 47kDa toxin from 167H2.

[0034] SEQ ID NO:7 is a 25-amino acid N-terminal sequence for thepurified approximately 14 kDa protein from PS167H2.

[0035] SEQ ID NO:8 is an oligonucleotide probe for the gene encoding thePS80JJ1 44.3 kDa toxin and is a forward primer for PS149B1 and PS167H2used according to the subject invention.

[0036] SEQ ID NO:9 is a reverse primer for PS149B1 and PS167H2 usedaccording to the subject invention.

[0037] SEQ ID NO:10 is the nucleotide sequence of the gene encoding theapproximately 45 kDa PS80JJ1 toxin.

[0038] SEQ ID NO:11 is the amino acid sequence for the approximately 45kDa PS80JJ1 toxin.

[0039] SEQ ID NO:12 is the partial nucleotide sequence of the geneencoding the approximately 44 kDa PS149B1 toxin.

[0040] SEQ ID NO:13 is the partial amino acid sequence for theapproximately 44 kDa PS149B1 toxin.

[0041] SEQ ID NO:14 is the partial nucleotide sequence of the geneencoding the approximately 44 kDa PS167H2 toxin.

[0042] SEQ ID NO:15 is the partial amino acid sequence for theapproximately 44 kDa PS167H2 toxin.

[0043] SEQ ID NO:16 is a peptide sequence used in primer designaccording to the subject invention.

[0044] SEQ ID NO:17 is a peptide sequence used in primer designaccording to the subject invention.

[0045] SEQ ID NO:18 is a peptide sequence used in primer designaccording to the subject invention.

[0046] SEQ ID NO:19 is a peptide sequence used in primer designaccording to the subject invention.

[0047] SEQ ID NO:20 is a nucleotide sequence corresponding to thepeptide of SEQ ID NO:16.

[0048] SEQ ID NO:21 is a nucleotide sequence corresponding to thepeptide of SEQ ID NO:17.

[0049] SEQ ID NO:22 is a nucleotide sequence corresponding to thepeptide of SEQ ID NO:18.

[0050] SEQ ID NO:23 is a nucleotide sequence corresponding to thepeptide of SEQ ID NO:19.

[0051] SEQ ID NO:24 is a reverse primer based on the reverse complementof SEQ ID NO:22.

[0052] SEQ ID NO:25 is a reverse primer based on the reverse complementof SEQ ID NO:23.

[0053] SEQ ID NO:26 is a forward primer based on the PS80JJ1 44.3 kDatoxin.

[0054] SEQ ID NO:27 is a reverse primer based on the PS80JJ1 44.3 kDatoxin.

[0055] SEQ ID NO:28 is a generic sequence representing a new class oftoxins according to the subject invention.

[0056] SEQ ID NO:29 is an oligonucleotide probe used according to thesubject invention.

[0057] SEQ ID NO:30 is the nucleotide sequence of the entire geneticlocus containing open reading frames of both the 14 and 45 kDa PS80JJ1toxins and the flanking nucleotide sequences.

[0058] SEQ ID NO:31 is the nucleotide sequence of the PS80JJ1 14 kDatoxin open reading frame.

[0059] SEQ ID NO:32 is the deduced amino acid sequence of the 14 kDatoxin of PS80JJ1.

[0060] SEQ ID NO:33 is a reverse oligonucleotide primer used accordingto the subject invention.

[0061] SEQ ID NO:34 is the nucleotide sequence of the entire geneticlocus containing open reading frames of both the 14 and 44 kDa PS167H2toxins and the flanking nucleotide sequences.

[0062] SEQ ID NO:35 is the nucleotide sequence of the gene encoding theapproximately 14 kDa PS167H2 toxin.

[0063] SEQ ID NO:36 is the amino acid sequence for the approximately 14kDa PS167H2 toxin.

[0064] SEQ ID NO:37 is the nucleotide sequence of the gene encoding theapproximately 44 kDa PS167H2 toxin.

[0065] SEQ ID NO:38 is the amino acid sequence for the approximately 44kDa PS167H2 toxin.

[0066] SEQ ID NO:39 is the nucleotide sequence of the entire geneticlocus containing open reading frames of both the 14 and 44 kDa PS149B1toxins and the flanking nucleotide sequences.

[0067] SEQ ID NO:40 is the nucleotide sequence of the gene encoding theapproximately 14 kDa PS149B1 toxin.

[0068] SEQ ID NO:41 is the amino acid sequence for the approximately 14kDa PS149B1 toxin.

[0069] SEQ ID NO:42 is the nucleotide sequence of the gene encoding theapproximately 44 kDa PS149B1 toxin.

[0070] SEQ ID NO:43 is the amino acid sequence for the approximately 44kDa PS149B1 toxin.

[0071] SEQ ID NO:44 is a maize-optimized gene sequence encoding theapproximately 14 kDa toxin of 80JJ1.

[0072] SEQ ID NO:45 is a maize-optimized gene sequence encoding theapproximately 44 kDa toxin of 80JJ1.

[0073] SEQ ID NO:46 is the DNA sequence of a reverse primer used inExample 15, below.

[0074] SEQ ID NO:47 is the DNA sequence of a forward primer (see Example16).

[0075] SEQ ID NO:48 is the DNA sequence of a reverse primer (see Example16).

[0076] SEQ ID NO:49 is the DNA sequence of a forward primer (see Example16).

[0077] SEQ ID NO:50 is the DNA sequence of a reverse primer (see Example16).

[0078] SEQ ID NO:51 is the DNA sequence from PS131W2 which encodes the14 kDa protein.

[0079] SEQ ID NO:52 is the amino acid sequence of the 14 kDa protein ofPS131W2.

[0080] SEQ ID NO:53 is a partial DNA sequence from PS131W2 for the 44kDa protein.

[0081] SEQ ID NO:54 is a partial amino acid sequence for the 44 kDaprotein of PS131W2.

[0082] SEQ ID NO:55 is the DNA sequence from PS158T3 which encodes the14 kDa protein.

[0083] SEQ ID NO:56 is the amino acid sequence of the 14 kDa protein ofPS158T3.

[0084] SEQ ID NO:57 is a partial DNA sequence from PS158T3 for the 44kDa protein.

[0085] SEQ ID NO:58 is a partial amino acid sequence for the 44 kDaprotein of PS158T3.

[0086] SEQ ID NO:59 is the DNA sequence from PS158X10 which encodes the14 kDa protein.

[0087] SEQ ID NO:60 is the amino acid sequence of the 14 kDa protein ofPS158X10.

[0088] SEQ ID NO:61 is the DNA sequence from PS185FF which encodes the14 kDa protein.

[0089] SEQ ID NO:62 is the amino acid sequence of the 14 kDa protein ofPS185FF.

[0090] SEQ ID NO:63 is a partial DNA sequence from PS185FF for the 44kDa protein.

[0091] SEQ ID NO:64 is a partial amino acid sequence for the 44 kDaprotein of PS185FF.

[0092] SEQ ID NO:65 is the DNA sequence from PS185GG which encodes the14 kDa protein.

[0093] SEQ ID NO:66 is the amino acid sequence of the 14 kDa protein ofPS185GG.

[0094] SEQ ID NO:67 is the DNA sequence from PS185GG for the 44 kDaprotein.

[0095] SEQ ID NO:68 is the amino acid sequence for the 44 kDa protein ofPS185GG.

[0096] SEQ ID NO:69 is the DNA sequence from PS185L12 which encodes the14 kDa protein.

[0097] SEQ ID NO:70 is the amino acid sequence of the 14 kDa protein ofPS185L12.

[0098] SEQ ID NO:71 is the DNA sequence from PS185W3 which encodes the14 kDa protein.

[0099] SEQ ID NO:72 is the amino acid sequence of the 14 kDa protein ofPS185W3.

[0100] SEQ ID NO:73 is the DNA sequence from PS186FF which encodes the14 kDa protein.

[0101] SEQ ID NO:74 is the amino acid sequence of the 14 kDa protein ofPS186FF.

[0102] SEQ ID NO:75 is the DNA sequence from PS187F3 which encodes the14 kDa protein.

[0103] SEQ ID NO:76 is the amino acid sequence of the 14 kDa protein ofPS187F3.

[0104] SEQ ID NO:77 is a partial DNA sequence from PS187F3 for the 44kDa protein.

[0105] SEQ ID NO:78 is a partial amino acid sequence for the 44 kDaprotein of PS187F3.

[0106] SEQ ID NO:79 is the DNA sequence from PS187G1 which encodes the14 kDa protein.

[0107] SEQ ID NO:80 is the amino acid sequence of the 14 kDa protein ofPS187G1.

[0108] SEQ ID NO:81 is a partial DNA sequence from PS187G1 for the 44kDa protein.

[0109] SEQ ID NO:82 is a partial amino acid sequence for the 44 kDaprotein of PS187G1.

[0110] SEQ ID NO:83 is the DNA sequence from PS187L14 which encodes the14 kDa protein.

[0111] SEQ ID NO:84 is the amino acid sequence of the 14 kDa protein ofPS187L14.

[0112] SEQ ID NO:85 is a partial DNA sequence from PS187L14 for the 44kDa protein.

[0113] SEQ ID NO:86 is a partial amino acid sequence for the 44 kDaprotein of PS187L14.

[0114] SEQ ID NO:87 is the DNA sequence from PS187Y2 which encodes the14 kDa protein.

[0115] SEQ ID NO:88 is the amino acid sequence of the 14 kDa protein ofPS187Y2.

[0116] SEQ ID NO:89 is a partial DNA sequence from PS187Y2 for the 44kDa protein.

[0117] SEQ ID NO:90 is a partial amino acid sequence for the 44 kDaprotein of PS187Y2.

[0118] SEQ ID NO:91 is the DNA sequence from PS201G which encodes the 14kDa protein.

[0119] SEQ ID NO:92 is the amino acid sequence of the 14 kDa protein ofPS201 G.

[0120] SEQ ID NO:93 is the DNA sequence from PS201HH which encodes the14 kDa protein.

[0121] SEQ ID NO:94 is the amino acid sequence of the 14 kDa protein ofPS201HH.

[0122] SEQ ID NO:95 is the DNA sequence from PS201L3 which encodes the14 kDa protein.

[0123] SEQ ID NO:96 is the amino acid sequence of the 14 kDa protein ofPS201L3.

[0124] SEQ ID NO:97 is the DNA sequence from PS204C3 which encodes the14 kDa protein.

[0125] SEQ ID NO:98 is the amino acid sequence of the 14 kDa protein ofPS204C3.

[0126] SEQ ID NO:99 is the DNA sequence from PS204G4 which encodes the14 kDa protein.

[0127] SEQ ID NO:100 is the amino acid sequence of the 14 kDa protein ofPS204G4.

[0128] SEQ ID NO:101 is the DNA sequence from PS204I11 which encodes the14 kDa protein.

[0129] SEQ ID NO:102 is the amino acid sequence of the 14 kDa protein ofPS204I11.

[0130] SEQ ID NO:103 is the DNA sequence from PS204J7 which encodes the14 kDa protein.

[0131] SEQ ID NO:104 is the amino acid sequence of the 14 kDa protein ofPS204J7.

[0132] SEQ ID NO:105 is the DNA sequence from PS236B6 which encodes the14 kDa protein.

[0133] SEQ ID NO:106 is the amino acid sequence of the 14 kDa protein ofPS236B6.

[0134] SEQ ID NO:107 is the DNA sequence from PS242K10 which encodes the14 kDa protein.

[0135] SEQ ID NO:108 is the amino acid sequence of the 14 kDa protein ofPS242K10.

[0136] SEQ ID NO:109 is a partial DNA sequence from PS242K10 for the 44kDa protein.

[0137] SEQ ID NO:110 is a partial amino acid sequence for the 44 kDaprotein of PS242K10.

[0138] SEQ ID NO:111 is the DNA sequence from PS246P42 which encodes the14 kDa protein.

[0139] SEQ ID NO:112 is the amino acid sequence of the 14 kDa protein ofPS246P42.

[0140] SEQ ID NO:113 is the DNA sequence from PS69Q which encodes the 14kDa protein.

[0141] SEQ ID NO:114 is the amino acid sequence of the 14 kDa protein ofPS69Q.

[0142] SEQ ID NO:115 is the DNA sequence from PS69Q for the 44 kDaprotein.

[0143] SEQ ID NO:116 is the amino acid sequence for the 44 kDa proteinof PS69Q.

[0144] SEQ ID NO:117 is the DNA sequence from KB54 which encodes the 14kDa protein.

[0145] SEQ ID NO:118 is the amino acid sequence of the 14 kDa protein ofKB54.

[0146] SEQ ID NO:119 is the DNA sequence from KR1209 which encodes the14 kDa protein.

[0147] SEQ ID NO:120 is the amino acid sequence of the 14 kDa protein ofKR1209.

[0148] SEQ ID NO:121 is the DNA sequence from KR1369 which encodes the14 kDa protein.

[0149] SEQ ID NO:122 is the amino acid sequence of the 14 kDa protein ofKR1369.

[0150] SEQ ID NO:123 is the DNA sequence from KR589 which encodes the 14kDa protein.

[0151] SEQ ID NO:124 is the amino acid sequence of the 14 kDa protein ofKR589.

[0152] SEQ ID NO:125 is a partial DNA sequence from KR589 for the 44 kDaprotein.

[0153] SEQ ID NO:126 is a partial amino acid sequence for the 44 kDaprotein of KR589.

[0154] SEQ ID NO:127 is a polynucleotide sequence for a gene designated149B1-15-PO, which is optimized for expression in Zea mays. This geneencodes an approximately 15 kDa toxin obtainable from PS149B1 that isdisclosed in WO 97/40162.

[0155] SEQ ID NO:128 is a polynucleotide sequence for a gene designated149B1-45-PO, which is optimized for expression in Zea mays. This geneencodes an approximately 45 kDa toxin obtainable from PS149B1 that isdisclosed in WO 97/40162.

[0156] SEQ ID NO:129 is a polynucleotide sequence for a gene designated80JJ1-15-PO7, which is optimized for expression in maize. This is analternative gene that encodes an approximately 15 kDa toxin.

[0157] SEQ ID NO:130 is an amino acid sequence for a toxin encoded bythe gene designated 80JJ1-15-PO7.

[0158] SEQ ID NO:131 is an oligonucleotide primer (15 kfor1) usedaccording to the subject invention (see Example 20).

[0159] SEQ ID NO:132 is an oligonucleotide primer (45krev6) usedaccording to the subject invention (see Example 20).

[0160] SEQ ID NO:133 is the DNA sequence from PS201L3 which encodes the14 kDa protein.

[0161] SEQ ID NO:134 is the amino acid sequence of the 14 kDa protein ofPS201 L3.

[0162] SEQ ID NO:135 is a DNA sequence from PS201L3 for the 44 kDaprotein.

[0163] SEQ ID NO:136 is an amino acid sequence for the 44 kDa protein ofPS201 L3.

[0164] SEQ ID NO:137 is the DNA sequence from PS187G1 which encodes the14 kDa protein.

[0165] SEQ ID NO:138 is the amino acid sequence of the 14 kDa protein ofPS187G1.

[0166] SEQ ID NO:139 is the DNA sequence from PS187G1 which encodes the44 kDa protein.

[0167] SEQ ID NO:140 is the amino acid sequence of the 44 kDa protein ofPS187G1.

[0168] SEQ ID NO:141 is the DNA sequence from PS201HH2 which encodes the14 kDa protein.

[0169] SEQ ID NO:142 is the amino acid sequence of the 14 kDa protein ofPS201HH2.

[0170] SEQ ID NO:143 is a partial DNA sequence from PS201HH2 for the 44kDa protein.

[0171] SEQ ID NO:144 is a partial amino acid sequence for the 44 kDaprotein of PS201HH2.

[0172] SEQ ID NO:145 is the DNA sequence from KR1369 which encodes the14 kDa protein.

[0173] SEQ ID NO:146 is the amino acid sequence of the 14 kDa protein ofKR1369.

[0174] SEQ ID NO:147 is the DNA sequence from KR1369 which encodes the44 kDa protein.

[0175] SEQ ID NO:148 is the amino acid sequence of the 44 kDa protein ofKR1369.

[0176] SEQ ID NO:149 is the DNA sequence from PS137A which encodes the14 kDa protein.

[0177] SEQ ID NO:150 is the amino acid sequence of the 14 kDa protein ofPS137A.

[0178] SEQ ID NO:151 is the DNA sequence from PS201V2 which encodes the14 kDa protein.

[0179] SEQ ID NO:152 is the amino acid sequence of the 14 kDa protein ofPS201V2.

[0180] SEQ ID NO:153 is the DNA sequence from PS207C3 which encodes the14 kDa protein.

[0181] SEQ ID NO:154 is the amino acid sequence of the 14 kDa protein ofPS207C3.

[0182] SEQ ID NO:155 is an oligonucleotide primer (F1new) for useaccording to the subject invention (see Example 22).

[0183] SEQ ID NO:156 is an oligonucleotide primer (R1new) for useaccording to the subject invention (see Example 22).

[0184] SEQ ID NO:157 is an oligonucleotide primer (F2new) for useaccording to the subject invention (see Example 22).

[0185] SEQ ID NO:158 is an oligonucleotide primer (R2new) for useaccording to the subject invention (see Example 22).

[0186] SEQ ID NO:159 is an approximately 58 kDa fusion protein.

[0187] SEQ ID NO:160 is a fusion gene encoding the protein of SEQ IDNO:159.

[0188] SEQ ID NO:161 is primer 45 kD5′ for use according to the subjectinvention (see Example 27).

[0189] SEQ ID NO:162 is primer 45 kD3′rc for use according to thesubject invention (see Example 27).

[0190] SEQ ID NO:163 is primer 45 kD5′01 for use according to thesubject invention (see Example 27).

[0191] SEQ ID NO:164 is primer 45 kD5′02 for use according to thesubject invention (see Example 27).

[0192] SEQ ID NO:165 is primer 45 kD3′03 for use according to thesubject invention (see Example 27).

[0193] SEQ ID NO:166 is primer 45 kD3′04 for use according to thesubject invention (see Example 27).

DETAILED DISCLOSURE OF THE INVENTION

[0194] The subject invention concerns two new classes of polynucleotidesequences as well as the novel pesticidal proteins encoded by thesepolynucleotides. In one embodiment, the proteins have a full-lengthmolecular weight of approximately 40-50 kDa. In specific embodimentsexemplified herein, these proteins have a molecular weight of about43-47 kDa. In a second embodiment, the pesticidal proteins have amolecular weight of approximately 10-15 kDa. In specific embodimentsexemplified herein, these proteins have a molecular weight of about13-14 kDa.

[0195] In preferred embodiments, a 40-50 kDa protein and a 10-15 kDaprotein are used together, and the proteins are pesticidal incombination. Thus, the two classes of proteins of the subject inventioncan be referred to as “binary toxins.” As used herein, the term “toxin”includes either class of pesticidal proteins. The subject inventionconcerns polynucleotides which encode either the 40-50 kDa or the 10-15kDa toxins, polynucleotides which encode portions or fragments of thefull length toxins that retain pesticidal activity when used incombination, and polynucleotide sequences which encode both types oftoxins. In a preferred embodiment, these toxins are active againstcoleopteran pests, more preferably corn rootworm, and most preferablyWestern corn rootworm. Lepidopteran pests can also be targeted.

[0196] Certain specific toxins are exemplified herein. For toxins havinga known amino acid sequence, the molecular weight is also known. Thoseskilled in the art will recognize that the apparent molecular weight ofa protein as determined by gel electrophoresis will sometimes differfrom the true molecular weight. Therefore, reference herein to, forexample, a 45 kDa protein or a 14 kDa protein is understood to refer toproteins of approximately that size even if the true molecular weight issomewhat different.

[0197] The subject invention concerns not only the polynucleotides thatencode these classes of toxins, but also the use of thesepolynucleotides to produce recombinant hosts which express the toxins.In a further aspect, the subject invention concerns the combined use ofan approximately 40-50 kDa toxin of the subject invention together withan approximately 10-15 kDa toxin of the subject invention to achievehighly effective control of pests, including coleopterans such as cornrootworm. For example, the roots of one plant can express both types oftoxins.

[0198] Thus, control of pests using the isolates, toxins, and genes ofthe subject invention can be accomplished by a variety of methods knownto those skilled in the art. These methods include, for example, theapplication of B.t. isolates to the pests (or their location), theapplication of recombinant microbes to the pests (or their locations),and the transformation of plants with genes which encode the pesticidaltoxins of the subject invention. Microbes for use according to thesubject invention maybe, for example, B.t., E. coli, and/or Pseudomonas.Recombinant hosts can be made by those skilled in the art using standardtechniques. Materials necessary for these transformations are disclosedherein or are otherwise readily available to the skilled artisan.Control of insects and other pests such as nematodes and mites can alsobe accomplished by those skilled in the art using standard techniquescombined with the teachings provided herein.

[0199] The new classes of toxins and polynucleotide sequences providedhere are defined according to several parameters. One criticalcharacteristic of the toxins described herein is pesticidal activity. Ina specific embodiment, these toxins have activity against coleopteranpests. Anti-lepidopteran-active toxins are also embodied. The toxins andgenes of the subject invention can be further defined by their aminoacid and nucleotide sequences. The sequences of the molecules withineach novel class can be identified and defined in terms of theirsimilarity or identity to certain exemplified sequences as well as interms of the ability to hybridize with, or be amplified by, certainexemplified probes and primers. The classes of toxins provided hereincan also be identified based on their immunoreactivity with certainantibodies and based upon their adherence to a generic formula.

[0200] It should be apparent to a person skilled in this art that genesencoding pesticidal proteins according to the subject invention can beobtained through several means. The specific genes exemplified hereinmay be obtained from the isolates deposited at a culture depository asdescribed herein. These genes, and toxins, of the subject invention canalso be constructed synthetically, for example, by the use of a genesynthesizer.

[0201] The sequence of three exemplary 45 kDa toxins are provided as SEQID NOS:11, 43, and 38. In preferred embodiments, toxins of this classhave a sequence which conforms to the generic sequence presented as SEQID NO:28. In preferred embodiments, the toxins of this class willconform to the consensus sequence shown in FIG. 1.

[0202] With the teachings provided herein, one skilled in the art couldreadily produce and use the various toxins and polynucleotide sequencesof the novel classes described herein.

[0203] Microorganisms useful according to the subject invention havebeen deposited in the permanent collection of the Agricultural ResearchService Patent Culture Collection (NRRL), Northern Regional ResearchCenter, 1815 North University Street, Peoria, Ill. 61604, USA. Theculture repository numbers of the deposited strains are as follows:Culture Repository No. Deposit Date B.t. strain PS80JJ1 NRRL B-18679Jul. 17, 1990 B.t. strain PS149B1 NRRL B-21553 Mar. 28, 1996 B.t. strainPS167H2 NRRL B-21554 Mar. 28, 1996 E. coli NM522 (pMYC2365) NRRL B-21170Jan. 5, 1994 E. coli NM522 (pMYC2382) NRRL B-21329 Sep. 28, 1994 E. coliNM522 (pMYC2379) NRRL B-21155 Nov. 3, 1993 E. coli NM522(pMYC2421) NRRLB-21555 Mar. 28, 1996 E. coli NM522(pMYC2427) NRRL B-21672 Mar. 26, 1997E. coli NM522(pMYC2429) NRRL B-21673 Mar. 26, 1997 E. coliNM522(pMYC2426) NRRL B-21671 Mar. 26, 1997 B.t. strain PS185GG NRRLB-30175 Aug. 19, 1999 B.t. strain PS187G1 NRRL B-30185 Aug. 19, 1999B.t. strain PS187Y2 NRRL B-30187 Aug. 19, 1999 B.t. strain PS201G NRRLB-30188 Aug. 19, 1999 B.t. strain PS201HH2 NRRL B-30190 Aug. 19, 1999B.t. strain PS242K10 NRRL B-30195 Aug. 19, 1999 B.t. strain PS69Q NRRLB-30175 Aug. 19, 1999 B.t. strain KB54A1-6 NRRL B-30197 Aug. 19, 1999B.t. strain KR589 NRRL B-30198 Aug. 19, 1999 B.t. strain PS185L12 NRRLB-30179 Aug. 19, 1999 B.t. strain PS185W3 NRRL B-30180 Aug. 19, 1999B.t. strain PS187L14 NRRL B-30186 Aug. 19, 1999 B.t. strain PS186FF NRRLB-30182 Aug. 19, 1999 B.t. strain PS131W2 NRRL B-30176 Aug. 19, 1999B.t. strain PS158T3 NRRL B-30177 Aug. 19, 1999 B.t. strain PS158X10 NRRLB-30178 Aug. 19, 1999 B.t. strain PS185FF NRRL B-30182 Aug. 19, 1999B.t. strain PS187F3 NRRL B-30184 Aug. 19, 1999 B.t. strain PS201L3 NRRLB-30189 Aug. 19, 1999 B.t. strain PS204C3 NRRL B-30191 Aug. 19, 1999B.t. strain PS204G4 NRRL B-18685 Jul. 17, 1990 B.t. strain PS204I11 NRRLB-30192 Aug. 19, 1999 B.t. strain PS204J7 NRRL B-30193 Aug. 19, 1999B.t. strain PS236B6 NRRL B-30194 Aug. 19, 1999 B.t. strain PS246P42 NRRLB-30196 Aug. 19, 1999 B.t. strain KR1209 NRRL B-30199 Aug. 19, 1999 B.t.strain KR1369 NRRL B-30200 Aug. 19, 1999 B.t. strain MR1506 NRRL B-30298June 1, 2000 B.t. strain MR1509 NRRL B-30330 Aug. 8, 2000 B.t. strainMR1510 NRRL B-30331 Aug. 8, 2000 P.f. strain MR1607 NRRL B-30332 Aug. 8,2000

[0204] The PS80JJ1 isolate is available to the public by virtue of theissuance of U.S. Pat. No. 5,151,363 and other patents.

[0205] A further aspect of the subject invention concerns novel isolatesand the toxins and genes obtainable from these isolates. Novel isolateshave been deposited and are included in the above list. These isolateshave been deposited under conditions that assure that access to thecultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0206] Further, the subject culture deposits will be stored and madeavailable to the public in accord with the provisions of the BudapestTreaty for the Deposit of Microorganisms, i.e., they will be stored withall the care necessary to keep them viable and uncontaminated for aperiod of at least five years after the most recent request for thefurnishing of a sample of a deposit, and in any case, for a period of atleast 30 (thirty) years after the date of deposit or for the enforceablelife of any patent which may issue disclosing the cultures. Thedepositor acknowledges the duty to replace the deposit(s) should thedepository be unable to furnish a sample when requested, due to thecondition of the deposit(s). All restrictions on the availability to thepublic of the subject culture deposits will be irrevocably removed uponthe granting of a patent disclosing them.

[0207] Following is a table which provides characteristics of certainB.t. isolates that are useful according to the subject invention. TABLE1 Description of B.t. strains toxic to coleopterans Crystal Approx. MWNRRL Deposit Culture Description (kDa) Serotype Deposit Date PS80JJ1multiple attached 130, 90, 47, 37, 14 4a4b, sotto B-18679 Jul. 7, 1990PS149B1 130, 47, 14 B-21553 Mar. 28, 1996 PS167H2 70, 47, 14 B-23554Mar. 28, 1996

[0208] Other isolates of the subject invention can also be characterizedin terms of the shape and location of toxin inclusions.

[0209] Toxins, genes, and probes. The polynucleotides of the subjectinvention can be used to form complete “genes” to encode proteins orpeptides in a desired host cell. For example, as the skilled artisanwould readily recognize, some of the polynucleotides in the attachedsequence listing are shown without stop codons. Also, the subjectpolynucleotides can be appropriately placed under the control of apromoter in a host of interest, as is readily known in the art.

[0210] As the skilled artisan would readily recognize, DNA typicallyexists in a double-stranded form. In this arrangement, one strand iscomplementary to the other strand and vice versa. As DNA is replicatedin a plant (for example) additional, complementary strands of DNA areproduced. The “coding strand” is often used in the art to refer to thestrand that binds with the anti-sense strand. The mRNA is transcribedfrom the “anti-sense” strand of DNA. The “sense” or “coding” strand hasa series of codons (a codon is three nucleotides that can be readthree-at-a-time to yield a particular amino acid) that can be read as anopen reading frame (ORF) to form a protein or peptide of interest. Inorder to express a protein in vivo, a strand of DNA is typicallytranscribed into a complementary strand of mRNA which is used as thetemplate for the protein. Thus, the subject invention includes the useof the exemplified polynucleotides shown in the attached sequencelisting and/or the complementary strands. RNA and PNA (peptide nucleicacids) that are functionally equivalent to the exemplified DNA areincluded in the subject invention.

[0211] Toxins and genes of the subject invention can be identified andobtained by using oligonucleotide probes, for example. These probes aredetectable nucleotide sequences which may be detectable by virtue of anappropriate label or may be made inherently fluorescent as described inInternational Application No. WO 93/16094. The probes (and thepolynucleotides of the subject invention) may be DNA, RNA, or PNA. Inaddition to adenine (A), cytosine (C), guanine (G), thymine (T), anduracil (U; for RNA molecules), synthetic probes (and polynucleotides) ofthe subject invention can also have inosine (a neutral base capable ofpairing with all four bases; sometimes used in place of a mixture of allfour bases in synthetic probes). Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “n” is used generically, “n”can be G, A, T, C, or inosine. Ambiguity codes as used herein are inaccordance with standard IUPAC naming conventions as of the filing ofthe subject application (for example, R means A or G, Y means C or T,etc.)

[0212] As is well known in the art, if the probe molecule and nucleicacid sample hybridize by forming a strong bond between the twomolecules, it can be reasonably assumed that the probe and sample havesubstantial homology/similarity/identity. Preferably, hybridization isconducted under stringent conditions by techniques well-known in theart, as described in, for example, Keller, G. H., M. M. Manak (11987)DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. For example, asstated therein, high stringency conditions can be achieved by firstwashing with 2×SSC (Standard Saline Citrate)/0.1% SDS (Sodium DodecylSulfate) for 15 minutes at room temperature. Two washes are typicallyperformed. Higher stringency can then be achieved by lowering the saltconcentration and/or by raising the temperature. For example, the washdescribed above can be followed by two washings with 0.1×SSC/0.1% SDSfor 15 minutes each at room temperature followed by subsequent washeswith 0.1×SSC/0.1% SDS for 30 minutes each at 55° C. These temperaturescan be used with other hybridization and wash protocols set forth hereinand as would be known to one skilled in the art (SSPE can be used as thesalt instead of SSC, for example). The 2×SSC/0.1% SDS can be prepared byadding 50 ml of 20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSCcan be prepared by combining NaCl (175.3 g/0.150 M), sodium citrate(88.2 g/0.015 M), and water to 1 liter, followed by adjusting pH to 7.0with 10 N NaOH. 10% SDS can be prepared by dissolving 10 g of SDS in 50ml of autoclaved water, diluting to 100 ml, and aliquotting.

[0213] Detection of the probe provides a means for determining in aknown manner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying toxin-encoding genes of thesubject invention. The nucleotide segments which are used as probesaccording to the invention can be synthesized using a DNA synthesizerand standard procedures. These nucleotide sequences can also be used asPCR primers to amplify genes of the subject invention.

[0214] Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide exemplifiedherein (such as the DNA sequences included in SEQ ID NOs:46-166).

[0215] As used herein “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots With ³²P-labeled gene-specific probes was performed by standardmethods (Maniatis, T., E. F. Fritsch, J. Sambrook [1982] MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). In general, hybridization and subsequent washes werecarried out under stringent conditions that allowed for detection oftarget sequences (with homology to the PS80JJ1 toxin genes, forexample). For double-stranded DNA gene probes, hybridization was carriedout overnight at 20-25° C. below the melting temperature (Tm) of the DNAhybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denaturedDNA. The melting temperature is described by the following formula(Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C.Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave[eds.] Academic Press, New York 100:266-285):

[0216] T_(m)=81.5° C.+16.6 Log[Na+]+0.41 (% G+C)-0.61 (%formamide)-600/length of duplex in base pairs.

[0217] Washes are typically carried out as follows:

[0218] (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS(low stringency wash).

[0219] (2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS(moderate stringency wash).

[0220] For oligonucleotide probes, hybridization was carried outovernight at 10-20° C. below the melting temperature (Tm) of the hybridin 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tmfor oligonucleotide probes was determined by the following formula:

[0221] Tm (° C.)=2 (number T/A base pairs)+4 (number G/C base pairs)

[0222] (Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K.Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. UsingPurified Genes, D. D. Brown [ed.], Academic Press, New York,23:683-693).

[0223] Washes were typically carried out as follows:

[0224] (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS(low stringency wash).

[0225] (2) Once at the hybridization temperature for 15 minutes in1×SSPE, 0.1% SDS (moderate stringency wash).

[0226] Toxins obtainable from isolates PS149B1, PS167H2, and PS80JJ1have been characterized as having have at least one of the followingcharacteristics (novel toxins of the subject invention can be similarlycharacterized with this and other identifying information set forthherein):

[0227] (a) said toxin is encoded by a nucleotide sequence whichhybridizes under stringent conditions with a nucleotide sequenceselected from the group consisting of: DNA which encodes SEQ ID NO:2,DNA which encodes SEQ ID NO:4, DNA which encodes SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, DNA which encodes SEQ ID NO:11, SEQ ID NO:12, DNAwhich encodes SEQ ID NO:13, SEQ ID NO:14, DNA which encodes SEQ IDNO:15, DNA which encodes SEQ ID NO:16, DNA which encodes SEQ ID NO:17,DNA which encodes SEQ ID NO:18, DNA which encodes SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, DNA which encodes a pesticidalportion of SEQ ID NO:28, SEQ ID NO:37, DNA which encodes SEQ ID NO:38,SEQ ID NO:42, and DNA which encodes SEQ ID NO:43;

[0228] (b) said toxin immunoreacts with an antibody to an approximately40-50 kDa pesticidal toxin, or a fragment thereof, from a Bacillusthuringiensis isolate selected from the group consisting of PS80JJ1having the identifying characteristics of NRRL B-18679, PS149B1 havingthe identifying characteristics of NRRL B-21553, and PS167H2 having theidentifying characteristics of NRRL B-21554;

[0229] (c) said toxin is encoded by a nucleotide sequence wherein aportion of said nucleotide sequence can be amplified by PCR using aprimer pair selected from the group consisting of SEQ ID NOs:20 and 24to produce a fragment of about 495 bp, SEQ ID NOs:20 and 25 to produce afragment of about 594 bp, SEQ ID NOs:21 and 24 to produce a fragment ofabout 471 bp, and SEQ ID NOs:21 and 25 to produce a fragment of about580 bp;

[0230] (d) said toxin comprises a pesticidal portion of the amino acidsequence shown in SEQ ID NO:28;

[0231] (e) said toxin comprises an amino acid sequence which has atleast about 60% homology with a pesticidal portion of an amino acidsequence selected from the group consisting of SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:38, and SEQ ID NO:43;

[0232] (f) said toxin is encoded by a nucleotide sequence whichhybridizes under stringent conditions with a nucleotide sequenceselected from the group consisting of DNA which encodes SEQ ID NO:3, DNAwhich encodes SEQ ID NO:5, DNA which encodes SEQ ID NO:7, DNA whichencodes SEQ ID NO:32, DNA which encodes SEQ ID NO:36, and DNA whichencodes SEQ ID NO:41;

[0233] (g) said toxin immunoreacts with an antibody to an approximately10-15 kDa pesticidal toxin, or a fragment thereof, from a Bacillusthuringiensis isolate selected from the group consisting of PS80JJ1having the identifying characteristics of NRRL B-18679, PS149B1 havingthe identifying characteristics of NRRL B-21553, and PS167H2 having theidentifying characteristics of NRRL B-21554;

[0234] (h) said toxin is encoded by a nucleotide sequence wherein aportion of said nucleotide sequence can be amplified by PCR using theprimer pair of SEQ ID NO:29 and SEQ ID NO:33; and

[0235] (i) said toxin comprises an amino acid sequence which has atleast about 60% homology with an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, pesticidalportions of SEQ ID NO:32, pesticidal portions of SEQ ID NO:36, andpesticidal portions of SEQ ID NO:41.

[0236] Modification of genes and toxins. The genes and toxins usefulaccording to the subject invention include not only the specificallyexemplified full-length sequences, but also portions and/or fragments(including internal and/or terminal deletions compared to thefull-length molecules) of these sequences, variants, mutants, chimerics,and fusions thereof. Proteins of the subject invention can havesubstituted amino acids so long as they retain the characteristicpesticidal activity of the proteins specifically exemplified herein.“Variant” genes have nucleotide sequences which encode the same toxinsor which encode toxins having pesticidal activity equivalent to anexemplified protein. As used herein, the term “equivalent toxins” refersto toxins having the same or essentially the same biological activityagainst the target pests as the exemplified toxins. As used herein,reference to “essentially the same” sequence refers to sequences whichhave amino acid substitutions, deletions, additions, or insertions whichdo not materially affect pesticidal activity. Fragments retainingpesticidal activity are also included in this definition. Fragments andequivalents which retain the pesticidal activity of the exemplifiedtoxins would be within the scope of the subject invention.

[0237] Equivalent toxins and/or genes encoding these equivalent toxinscan be derived from wild-type or recombinant B.t. isolates and/or fromother wild-type or recombinant organisms using the teachings providedherein. Other Bacillus species, for example, can be used as sourceisolates.

[0238] Variations of genes may be readily constructed using standardtechniques for making point mutations, for example. Also, U.S. Pat. No.5,605,793, for example, describes methods for generating additionalmolecular diversity by using DNA reassembly after random fragmentation.Variant genes can be used to produce variant proteins; recombinant hostscan be used to produce the variant proteins. Fragments of full-lengthgenes can be made using commercially available exonucleases orendonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, genes whichencode active fragments may be obtained using a variety of restrictionenzymes. Proteases may be used to directly obtain active fragments ofthese toxins.

[0239] There are a number of methods for obtaining the pesticidal toxinsof the instant invention. For example, antibodies to the pesticidaltoxins disclosed and claimed herein can be used to identify and isolateother toxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the toxins which are most constant and mostdistinct from other B.t. toxins. These antibodies can then be used tospecifically identify equivalent toxins with the characteristic activityby immunoprecipitation, enzyme linked immunosorbent assay (ELISA), orwestern blotting. Antibodies to the toxins disclosed herein, or toequivalent toxins, or to fragments of these toxins, can readily beprepared using standard procedures. The genes which encode these toxinscan then be obtained from the source microorganism.

[0240] Because of the redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate these alternative DNA sequences encoding the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention.

[0241] Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid similarity (and/or homology) with an exemplified toxin.The amino acid identity will typically be greater than 60%, preferablygreater than 75%, more preferably greater than 80%, even more preferablygreater than 90%, and can be greater than 95%. Preferred polynucleotidesand proteins of the subject invention can also be defined in terms ofmore particular identity and/or similarity ranges. For example, theidentity and/or similarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.Unless otherwise specified, as used herein percent sequence identityand/or similarity of two nucleic acids is determined using the algorithmof Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215:402-410.BLAST nucleotide searches are performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences with thedesired percent sequence identity. To obtain gapped aligrnents forcomparison purposes, Gapped BLAST is used as described in Altschul etal. (1997), Nucl. Acids Res. 25:3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) are used. See http://www.ncbi.nih.gov. The scorescan also be calculated using the methods and algorithms of Crickmore etal. as described in the Background section, above.

[0242] The amino acid homology will be highest in critical regions ofthe toxin which account for biological activity or are involved in thedetermination of three-dimensional configuration which ultimately isresponsible for the biological activity. In this regard, certain aminoacid substitutions are acceptable and can be expected if thesesubstitutions are in regions which are not critical to activity or areconservative amino acid substitutions which do not affect thethree-dimensional configuration of the molecule. For example, aminoacids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 2 provides a listing of examples of amino acidsbelonging to each class. TABLE 2 Class of Amino Acid Examples of AminoAcids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged PolarGly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

[0243] In some instances, non-conservative substitutions can also bemade. The critical factor is that these substitutions must notsignificantly detract from the biological activity of the toxin.

[0244] As used herein, reference to “isolated” polynucleotides and/or“purified” toxins refers to these molecules when they are not associatedwith the other molecules with which they would be found in nature; theseterms would include their use in plants. Thus, reference to “isolated”and/or “purified” signifies the involvement of the “hand of man” asdescribed herein.

[0245] Synthetic genes which are functionally equivalent to the toxinsof the subject invention can also be used to transform hosts. Methodsfor the production of synthetic genes can be found in, for example, U.S.Pat. No. 5,380,831.

[0246] Transgenic hosts. The toxin-encoding genes of the subjectinvention can be introduced into a wide variety of microbial or planthosts. In preferred embodiments, expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide proteins. When transgenic/recombinant/transformed hostcells are ingested by the pests, the pests will ingest the toxin. Thisis the preferred manner in which to cause contact of the pest with thetoxin. The result is a control (killing or making sick) of the pest.Alternatively, suitable microbial hosts, e.g., Pseudomonas such as P.fluorescens, can be applied to the situs of the pest, where some ofwhich can proliferate, and are ingested by the target pests. The microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin and stabilize the cell. The treated cell, whichretains the toxic activity, then can be applied to the environment ofthe target pest.

[0247] In preferred embodiments, recombinant plant cells and plants areused. Preferred plants (and plant cells) are corn and/or maize.

[0248] Where the B.t. toxin gene is introduced via a suitable vectorinto a microbial host, and said host is applied to the environment in aliving state, certain host microbes should be used. Microorganism hostsare selected which are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops ofinterest. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

[0249] A large number of microorganisms are known to inhabit thephylloplane (the surface of the plant leaves) and/or the rhizosphere(the soil surrounding plant roots) of a wide variety of important crops.These microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

[0250] A wide variety of ways are available for introducing a B.t. geneencoding a toxin into the target host under conditions which allow forstable maintenance and expression of the gene. These methods are wellknown to those skilled in the art and are described, for example, inU.S. Pat. No. 5,135,867, which is incorporated herein by reference.

[0251] Treatment of cells. As mentioned above, B.t. or recombinant cellsexpressing a B.t. toxin can be treated to prolong the toxin activity andstabilize the cell. The pesticide microcapsule that is formed comprisesthe B.t. toxin within a cellular structure that has been stabilized andwill protect the toxin when the microcapsule is applied to theenvironment of the target pest. Suitable host cells may include eitherprokaryotes or eukaryotes, normally being limited to those cells whichdo not produce substances toxic to higher organisms, such as mammals.However, organisms which produce substances toxic to higher organismscould be used, where the toxic substances are unstable or the level ofapplication sufficiently low as to avoid any possibility of toxicity toa mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi.

[0252] The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

[0253] Treatment of the microbial cell, e.g., a microbe containing theB.t. toxin gene, can be by chemical or physical means, or by acombination of chemical and/or physical means, so long as the techniquedoes not deleteriously affect the properties of the toxin, nor diminishthe cellular capability of protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as glutaraldehyde;anti-infectives, such as zephiran chloride and cetylpyridinium chloride;alcohols, such as isopropyl and ethanol; various histologic fixatives,such as Lugol iodine, Bouin's fixative, various acids and Helly'sfixative (See: Humason, Gretchen L., Animal Tissue Techniques, W.H.Freeman and Company, 1967); or a combination of physical (heat) andchemical agents that preserve and prolong the activity of the toxinproduced in the cell when the cell is administered to the hostenvironment. Examples of physical means are short wavelength radiationsuch as gamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. Methods for treatment of microbial cellsare disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

[0254] The cells generally will have enhanced structural stability whichwill enhance resistance to environmental conditions. Where the pesticideis in a proform, the method of cell treatment should be selected so asnot to inhibit processing of the proform to the mature form of thepesticide by the target pest pathogen. For example, formaldehyde willcrosslink proteins and could inhibit processing of the proform of apolypeptide pesticide. The method of treatment should retain at least asubstantial portion of the bio-availability or bioactivity of the toxin.

[0255] Characteristics of particular interest in selecting a host cellfor purposes of production include ease of introducing the B.t. geneinto the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide microcapsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; survival in aqueous environments; lack ofmammalian toxicity; attractiveness to pests for ingestion; ease ofkilling and fixing without damage to the toxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

[0256] Growth of cells. The cellular host containing the B.t.insecticidal gene may be grown in any convenient nutrient medium,preferably where the DNA construct provides a selective advantage,providing for a selective medium so that substantially all or all of thecells retain the B.t. gene. These cells may then be harvested inaccordance with conventional ways. Alternatively, the cells can betreated prior to harvesting.

[0257] The B.t. cells of the invention can be cultured using standardart media and fermentation techniques. Upon completion of thefermentation cycle the bacteria can be harvested by first separating theB.t. spores and crystals from the fermentation broth by means well knownin the art. The recovered B.t. spores and crystals can be formulatedinto a wettable powder, liquid concentrate, granules or otherformulations by the addition of surfactants, dispersants, inertcarriers, and other components to facilitate handling and applicationfor particular target pests. These formulations and applicationprocedures are all well known in the art.

[0258] Formulations. Formulated bait granules containing an attractantand spores and crystals of the B.t. isolates, or recombinant microbescomprising the genes obtainable from the B.t. isolates disclosed herein,can be applied to the soil. Formulated product can also be applied as aseed-coating or root treatment or total plant treatment at later stagesof the crop cycle. Plant and soil treatments of B.t. cells may beemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

[0259] As would be appreciated by a person skilled in the art, thepesticidal concentration will vary widely depending upon the nature ofthe particular formulation, particularly whether it is a concentrate orto be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

[0260] The formulations can be applied to the environment of the pest,e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.

[0261] Mutants. Mutants of the isolates of the invention can be made byprocedures well known in the art. For example, an asporogenous mutantcan be obtained through ethylmethane sulfonate (EMS) mutagenesis of anisolate. The mutants can be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

[0262] A smaller percentage of the asporogenous mutants will remainintact and not lyse for extended fermentation periods; these strains aredesignated lysis minus (−). Lysis minus strains can be identified byscreening asporogenous mutants in shake flask media and selecting thosemutants that are still intact and contain toxin crystals at the end ofthe fermentation. Lysis minus strains are suitable for a cell treatmentprocess that will yield a protected, encapsulated toxin protein.

[0263] To prepare a phage resistant variant of said asporogenous mutant,an aliquot of the phage lysate is spread onto nutrient agar and allowedto dry. An aliquot of the phage sensitive bacterial strain is thenplated directly over the dried lysate and allowed to dry. The plates areincubated at 30° C. The plates are incubated for 2 days and, at thattime, numerous colonies could be seen growing on the agar. Some of thesecolonies are picked and subcultured onto nutrient agar plates. Theseapparent resistant cultures are tested for resistance by cross streakingwith the phage lysate. A line of the phage lysate is streaked on theplate and allowed to dry. The presumptive resistant cultures are thenstreaked across the phage line. Resistant bacterial cultures show nolysis anywhere in the streak across the phage line after overnightincubation at 30° C. The resistance to phage is then reconfirmed byplating a lawn of the resistant culture onto a nutrient agar plate. Thesensitive strain is also plated in the same manner to serve as thepositive control. After drying, a drop of the phage lysate is placed inthe center of the plate and allowed to dry. Resistant cultures showed nolysis in the area where the phage lysate has been placed afterincubation at 30° C. for 24 hours.

[0264] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Culturinz of B.t. Isolates of the Invention

[0265] A subculture of the B.t. isolates, or mutants thereof, can beused to inoculate the following medium, a peptone, glucose, saltsmedium. Bacto Peptone  7.5 g/l Glucose  1.0 g/l KH₂PO₄  3.4 g/l K₂HPO₄4.35 g/l Salt Solution  5.0 ml/l CaCl₂ Solution  5.0 ml/l pH  7.2

[0266] Salts Solution (100 ml) MgSO₄.7H₂O 2.46 g MnSO₄.H₂O 0.04 gZnSO₄.7H₂O 0.28 g FeSO₄.7H₂O 0.40 g

[0267] CaCl₂ Solution (100 ml) CaCl₂.2H₂O 3.66 g

[0268] The salts solution and CaCl₂ solution are filter-sterilized andadded to the autoclaved and cooked broth at the time of inoculation.Flasks are incubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

[0269] The above procedure can be readily scaled up to large fermentorsby procedures well known in the art.

[0270] The B.t. spores and/or crystals, obtained in the abovefermentation, can be isolated by procedures well known in the art. Afrequently-used procedure is to subject the harvested fermentation brothto separation techniques, e.g., centrifugation.

Example 2 Activity of Sporulated Bacillus thuringiensis Cultures on CornRootworm

[0271] Liquid cultures of PS80JJ1, PS149B1 or PS167H2 were grown tosporulation in shake flasks and pelleted by centrifugation. Culturepellets were resuspended in water and assayed for activity against cornrootworm in top load bioassays as described above. The amounts of 14 kDaand 44.3 kDa proteins present in the culture pellets were estimated bydensitometry and used to calculate specific activity expressed as LC₅₀.Activity of each native B. thuringiensis strain is presented in Table 3(WCRW top load bioassay of B.t. strains). TABLE 3 WCRW Top Load Bioassayof B.t. Strains B.t. strain LC₅₀ (μg/cm²)* 95% CL Slope PS80JJ1 6 4-81.5 PS167H2 6 4-9 1.6 PS149B1 8 4-12 1.8 CryB cell blank 4% N/A N/AWater blank 4% N/A N/A

Example 3 Protein Purification for 45 kDa 80JJ1 Protein

[0272] One gram of lyophilized powder of 80JJ 1 was suspended in 40 mlof buffer containing 80 mM Tris-Cl pH 7.8, 5 mM EDTA, 100 μM PMSF, 0.5μg/ml Leupeptin, 0.7. μg/ml Pepstatin, and 40 μg/ml Bestatin. Thesuspension was centrifuged, and the resulting supernatant was discarded.The pellet was washed five times using 35-40 ml of the above buffer foreach wash. The washed pellet was resuspended in 10 ml of 40% NaBr, 5 mMEDTA, 100 μM PMSF, 0.5 μg/ml Leupeptin, 0.7 μg/ml Pepstatin, and 40μg/ml Bestatin and placed on a rocker platform for 75 minutes. The NaBrsuspension was centrifuged, the supernatant was removed, and the pelletwas treated a second time with 40% NaBr, 5 mM EDTA, 100 μM PMSF, 0.5μg/ml Leupeptin, 0.7 μg/ml Pepstatin, and 40 μg/ml Bestatin as above.The supernatants (40% NaBr soluble) were combined and dialyzed against10 mM CAPS pH 10.0, 1 mM EDTA at 4° C. The dialyzed extracts werecentrifuged and the resulting supernatant was removed. The pellet (40%NaBr dialysis pellet) was suspended in 5 ml of H₂O and centrifuged. Theresultant supernatant was removed and discarded. The washed pellet waswashed a second time in 10 ml of H₂O and centrifuged as above. Thewashed pellet was suspended in 1.5 ml of H₂O and contained primarilythree protein bands with apparent mobilities of approximately 47 kDa, 45kDa, and 15 kDa when analyzed using SDS-PAGE. At this stage ofpurification, the suspended 40% NaBr dialysis pellet containedapproximately 21 mg/ml of protein by Lowry assay.

[0273] The proteins in the pellet suspension were separated usingSDS-PAGE (Laemlli, U.K. [1970] Nature 227:680) in 15% acrylamide gels.The separated proteins were then electrophoretically blotted to a PVDFmembrane (Millipore Corp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 Vconstant. After one hour the PVDF membrane was rinsed in water brieflyand placed for 3 minutes in 0.25% Coomassie blue R-250, 50% methanol, 5%acetic acid. The stained membrane was destained in 40% MeOH, 5% aceticacid. The destained membrane was air-dried at room temperature (LeGendreet al. [1989] In A Practical Guide to Protein Purification ForMicrosequencing, P. Matsudaira, ed., Academic Press, New York, N.Y.).The membrane was sequenced using automated gas phase Edman degradation(Hunkapillar, M. W., R. M. Hewick, W. L. Dreyer, L. E. Hood [1983] Meth.Enzymol. 91:399).

[0274] The amino acid analysis revealed that the N-terminal sequence ofthe 45 kDa band was as follows: Met-Leu-Asp-Thr-Asn (SEQ ID NO:1).

[0275] The 47 kDa band was also analyzed and the N-terminal amino acidsequence was determined to be the same 5-amino acid sequence as SEQ IDNO:1. Therefore, the N-terminal amino acid sequences of the 47 kDapeptide and the 45 kDa peptide were identical.

[0276] This amino acid sequence also corresponds to a sequence obtainedfrom a 45 kDa peptide obtained from PS80JJ1 spore/crystal powders, usinganother purification protocol, with the N-terminal sequence as follows:Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-Ile-Ser-Asn-Leu-Ala-Asn-Gly-Leu-Tyr-Thr-Ser-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO:2).

Example 4 Purification of the 14 kDa Peptide of PS80JJ1

[0277] 0.8 ml of the white dialysis suspension (approximately 21 mg/ml)containing the 47 kDa, 45 kDa, and 15 kDa peptides, was dissolved in 10ml of 40% NaBr, and 0.5 ml of 100 mM EDTA were added. After about 18hours (overnight), a white opaque suspension was obtained. This wascollected by centrifugation and discarded. The supernatant wasconcentrated in a Centricon-30 (Amicon Corporation) to a final volume ofapproximately 15 ml. The filtered volume was washed with water by filterdialysis and incubated on ice, eventually forming a milky whitesuspension. The suspension was centrifuged and the pellet andsupernatant were separated and retained. The pellet was then suspendedin 1.0 ml water (approximately 6 mg/ml). The pellet containedsubstantially pure 15 kDa protein when analyzed by SDS-PAGE.

[0278] The N-terminal amino acid sequence was determined to be:Ser-Ala-Arg-Glu-Val-His-Ile-Glu-Ile-Asn-Asn-Thr-Arg-His-Thr-Leu-Gln-Leu-Glu-Ala-Lys-Thr-Lys-Leu(SEQ ID NO:3).

Example 5 Bioassay of Protein

[0279] A preparation of the insoluble fraction from the dialyzed NaBrextract of 80JJ1 containing the 47 kDa, 45 kDa, and 15 kDa peptides wasbioassayed against Western corn rootworm and were found to exhibitsignificant toxin activity.

Example 6 Protein Purification and Characterization of PS149B1 45 kDaProtein

[0280] The P1 pellet was resuspended with two volumes of deionized waterper unit wet weight, and to this was added nine volumes of 40% (w/w)aqueous sodium bromide. This and all subsequent operations were carriedout on ice or at 4-6° C. After 30 minutes, the suspension was dilutedwith 36 volumes of chilled water and centrifuged at 25,000×g for 30minutes to give a pellet and a supernatant.

[0281] The resulting pellet was resuspended in 1-2 volumes of water andlayered on a 20-40% (w/w) sodium bromide gradient and centrifuged at8,000×g for 100 minutes. The layer banding at approximately 32% (w/w)sodium bromide (the “inclusions”, or INC) was recovered and dialyzedovernight against water using a dialysis membrane with a 6-8 kDa MWcut-off. Particulate material was recovered by centrifugation at25,000×g, resuspended in water, and aliquoted and assayed for protein bythe method of Lowry and by SDS-PAGE.

[0282] The resulting supernatant was concentrated 3- to 4-fold usingCentricon-10 concentrators, then dialyzed overnight against water usinga dialysis membrane with a 6-8 kDa MW cut-off. Particulate material wasrecovered by centrifugation at 25,000×g, resuspended in water, andaliquoted and assayed for protein by the method of Lowry and bySDS-PAGE. This fraction was denoted as P1.P2.

[0283] The peptides in the pellet suspension were separated usingSDS-PAGE (Laemlli, U.K., supra) in 15% acrylamide gels. The separatedproteins were then electrophoretically blotted to a PVDF membrane(Millipore Corp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 V constant.After one hour the PVDF membrane was rinsed in water briefly and placedfor 3 minutes in 0.25% Coomassie blue R-250, 50% methanol, 5% aceticacid. The stained membrane was destained in 40% MeOH, 5% acetic acid.The destained membrane was air-dried at room temperature (LeGendre etal., supra). The membrane was sequenced using automated gas phase Edmandegradation (Hunkapillar et al., supra).

[0284] Protein analysis indicated the presence of two majorpolypeptides, with molecular weights of 47 kDa and 14 kDa. Molecularweights were measured against standard polypeptides of known molecularweight. This process provides only an estimate of true molecular weight.The 47 kDa band from PS149B1 migrated on SDS-PAGE in a mannerindistinguishable from the 47 kDa protein from PS80 μl. Likewise, the 14kDa band from PS149B1 migrated on SDS-PAGE in a manner indistinguishablefrom 14 kDa bands from PS167H2 and PS80JJ1. Apart from these twopolypeptides, which were estimated to account for 25-35% (47 kDa) and35-55% (15 kDa) of the Coomassie staining material respectively, theremay be minor bands, including those of estimated MW at 46 kDa, 130 kDa,and 70 kDa.

[0285] Protein analysis indicated that fraction INC contained a singlepolypeptide with MW of 47 kDa, and that fraction P1.P2 contained asingle polypeptide with MW of 14 kDa. These polypeptides were recoveredin yields greater than 50% from P1.

[0286] The N-terminal amino acid sequence for the purified 47 kDaprotein from PS149B1 is:Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-Ile-Ser-Asn-His-Ala-Asn-Gly-Leu-Tyr-Ala-Ala-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO:4).

[0287] The N-terminal amino acid sequence for the purified 14 kDaprotein from PS149B1 is:Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp-Lys-Thr-Lys-Leu-Asp-Gly-Gly-Arg-Trp-Arg-Thr-Ser-Pro-Xaa-Asn-Val-Ala-Asn-Asp-Gln-Ile-Lys-Thr-Phe-Val-Ala-Glu-Ser-Asn(SEQ ID NO:5).

Example 7 Amino Acid Sequence for 45 kDa and 14 kDa Toxins of PS167H2

[0288] The N-terminal amino acid sequence for the purified 45 kDaprotein from PS167H2 is:Met-Leu-Asp-Thr-Asn-Lys-Ile-Tyr-Glu-Ile-Ser-Asn-Tyr-Ala-Asn-Gly-Leu-His-Ala-Ala-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO:6).

[0289] The N-terminal amino acid sequence for the purified 14 kDaprotein from PS167H2 is:Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp-Lys-Thr-Lys-Leu(SEQ ID NO:7).

[0290] These amino acid sequences can be compared to the sequenceobtained for the 47 kDa peptide obtained from 80JJ1 spore/crystalpowders with the N-terminal sequence (SEQ ID NO:1) and to the sequenceobtained for the 14 kDa peptide obtained from 80JJ1 spore/crystalpowders with the N-terminal sequence (SEQ ID NO:3).

[0291] Clearly, the 45-47 kDa proteins are highly related, and the 14kDa proteins are highly related.

Example 8 Bioassay of Protein

[0292] The purified protein fractions from PS149B1 were bioassayedagainst western corn rootworm and found to exhibit significant toxinactivity when combined. In fact, the combination restored activity tothat noted in the original preparation (P1). The following bioassay dataset presents percent mortality and demonstrates this effect. TABLE 4Concentration (μg/cm²) P1 INC P1 · P2 INC + P1 · P2 300 88, 100, 94 1913 100 100 94, 50, 63 31 38 94 33.3 19, 19, 44 38 13 50 11.1 13, 56, 2512 31 13 3.7 0, 50, 0 0 31 13 1.2 13, 43, 12 0 12 19 0.4 6, 12, 6 25 196

Example 9 Molecular Cloning, Expression, and DNA Sequence Analysis of aNovel 6-Endotoxin Gene from Bacillus thuringiensis Strain PS80JJ1

[0293] Total cellular DNA was prepared from Bacillus thuringiensis(B.t.) cells grown to an optical density, at 600 nm, of 1.0. Cells werepelleted by centrifugation and resuspended in protoplast buffer (20mg/ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA).After incubation at 37° C. for 1 hour, protoplasts were lysed by twocycles of freezing and thawing. Nine volumes of a solution of 0.1 MNaCl, 0.1% SDS, 0.1 M Tris-Cl were added to complete lysis. The clearedlysate was extracted twice with phenol:chloroform (1:1). Nucleic acidswere precipitated with two volumes of ethanol and pelleted bycentrifugation. The pellet was resuspended in TE buffer and RNase wasadded to a final concentration of 50 μg/ml. After incubation at 37° C.for 1 hour, the solution was extracted once each with phenol:chloroform(1:1) and TE-saturated chloroform. DNA was precipitated from the aqueousphase by the addition of one-tenth volume of 3 M NaOAc and two volumesof ethanol. DNA was pelleted by centrifugation, washed with 70% ethanol,dried, and resuspended in TE buffer.

[0294] An oligonucleotide probe for the gene encoding the PS80JJ1 45 kDatoxin was designed from N-terminal peptide sequence data. The sequenceof the 29-base oligonucleotide probe was:

[0295] 5′-ATG YTW GAT ACW AAT AAA GTW TAT GAA AT-3′ (SEQ ID NO:8)

[0296] This oligonucleotide was mixed at four positions as shown. Thisprobe was radiolabeled with ³²P and used in standard conditionhybridization of Southern blots of PS80JJ1 total cellular DNA digestedwith various restriction endonucleases. Representative autoradiographicdata from these experiments showing the sizes of DNA restrictionfragments containing sequence homology to the 44.3 kDa toxinoligonucleotide probe of SEQ ID NO:8 are presented in Table 5. TABLE 5RFLP of PS80JJ1 cellular DNA fragments on Southern blots that hybridizedunder standard conditions with the 44.3 kDa toxin gene oligonucleotideprobe (SEQ ID NO: 8) Restriction Enzyme Approximate Fragment Size (kbp)EcoRI 6.0 HindIII 8.3 KpnI 7.4 PstI 11.5 XbaI 9.1

[0297] These DNA fragments identified in these analyses contain all or asegment of the PS80JJ1 45 kDa toxin gene. The approximate sizes of thehybridizing DNA fragments in Table 5 are in reasonable agreement withthe sizes of a subset of the PS80JJ1 fragments hybridizing with aPS80JJ1 45 kDa toxin subgene probe used in separate experiments, aspredicted (see Table 6, below).

[0298] A gene library was constructed from PS80JJ1 DNA partiallydigested with Sau3AI. Partial restriction digests were fractionated byagarose gel electrophoresis. DNA fragments 9.3 to 23 kbp in size wereexcised from the gel, electroeluted from the gel slice, purified on anElutip-D ion exchange column (Schleicher and Schuell, Keene, N. H.), andrecovered by ethanol precipitation. The Sau3AI inserts were ligated intoBamHI-digested LambdaGem-11 (Promega, Madison, Wis.). Recombinant phagewere packaged and plated on E. coli KW251 cells. Plaques were screenedby hybridization with the oligonucleotide probe described above.Hybridizing phage were plaque-purified and used to infect liquidcultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

[0299] Southern blot analysis revealed that one of the recombinant phageisolates contained an approximately 4.8 kbp XbaI-SacI band thathybridized to the PS80JJ1 toxin gene probe. The SacI site flanking thePS80JJ1 toxin gene is a phage vector cloning site, while the flankingXbaI site is located within the PS80JJ1 DNA insert. This DNA restrictionfragment was subcloned by standard methods into pBluescript S/K(Stratagene, San Diego, Calif.) for sequence analysis. The resultantplasmid was designated pMYC2421. The DNA insert was also subcloned intopHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised ofpBluescript S/K [Stratagene, La Jolla, Calif.] and the replicationorigin from a resident B.t. plasmid [D. Lereclus et al. (1989) FEMSMicrobiology Letters 60:211-218]) to yield pMYC2420.

[0300] An oligonucleotide probe for the gene encoding the PS80JJ1 14 kDatoxin was designed from N-terminal peptide sequence data. The sequenceof the 28-base oligonucleotide probe was: 5′ GW GAA GTW CAT ATW GAA ATWAAT AAT AC 3′ (SEQ ID NO:29). This oligonucleotide was mixed at fourpositions as shown. The probe was radiolabelled with ³²P and used instandard condition hybridizations of Southern blots of PS80JJ1 totalcellular and pMYC2421 DNA digested with various restrictionendonucleases. These RFLP mapping experiments demonstrated that the geneencoding the 14 kDa toxin is located on the same genomic EcoRI fragmentthat contains the N-terminal coding sequence for the 44.3 kDa toxin.

[0301] To test expression of the PS80JJ1 toxin genes in B.t., pMYC2420was transformed into the acrystalliferous (Cry-) B.t. host, CryB (A.Aronson, Purdue University, West Lafayette, Ind.), by electroporation.Expression of both the approximately 14 and 44.3 kDa PS80JJ1 toxinsencoded by pMYC2420 was demonstrated by SDS-PAGE analysis. Toxin crystalpreparations from the recombinant CryB[pMYC2420] strain, MR536, wereassayed and found to be active against western corn rootworm.

[0302] The PS80JJ1 toxin genes encoded by pMYC2421 were sequenced usingthe ABI373 automated sequencing system and associated software. Thesequence of the entire genetic locus containing both open reading framesand flanking nucleotide sequences is shown in SEQ ID NO:30. Thetermination codon of the 14 kDa toxin gene is 121 base pairs upstream(5′) from the initiation codon of the 44.3 kDa toxin gene (FIG. 2). ThePS80JJ1 14 kDa toxin open reading frame nucleotide sequence (SEQ IDNO:31), the 44.3 kDa toxin open reading frame nucleotide sequence (SEQID NO:10), and the respective deduced amino acid sequences (SEQ ID NO:32and SEQ ID NO:11) are novel compared to other toxin genes encodingpesticidal proteins.

[0303] Thus, the nucleotide sequence encoding the 14 kDa toxin ofPS80JJ1 is shown in SEQ ID NO:31. The deduced amino acid sequence of the14 kDa toxin of PS80JJ1 is shown in SEQ ID NO:32. The nucleotidesequences encoding both the 14 and 45 kDa toxins of PS80JJ1, as well asthe flanking sequences, are shown in SEQ ID NO:30. The relationship ofthese sequences is shown in FIG. 2.

[0304] A subculture of E. coli NM522 containing plasmid pMYC2421 wasdeposited in the permanent collection of the Patent Culture Collection(NRRL), Regional Research Center, 1815 North University Street, Peoria,Ill. 61604 USA on Mar. 28, 1996. The accession number is NRRL B-21555.

Example 10 RFLP and PCR Analysis of Additional Novel δ-Endotoxin Genesfrom Bacillus thuringiensis Strains PS149B1 and PS167H2

[0305] Two additional strains active against corn rootworm, PS149B1 andPS167H2, also produce parasporal protein crystals comprised in part ofpolypeptides approximately 14 and 45 kDa in size. Southern hybridizationand partial DNA sequence analysis were used to examine the relatednessof these toxins to the 80JJ1 toxins. DNA was extracted from these B.t,strains as described above, and standard Southern hybridizations wereperformed using the 14 kDa toxin oligonucleotide probe (SEQ ID NO:29)and an approximately 800 bp PCR fragment of the 80JJ1 44.3 kDa toxingene-encoding sequence. RFLP data from these experiments showing thesizes of DNA restriction fragments containing sequence homology to the44.3 kDa toxin are presented in Table 6. RFLP data from theseexperiments showing the sizes of DNA restriction fragments containingsequence homology to the approximately 14 kDa toxin are presented inTable 7. TABLE 6 RFLP of PS80JJ1, PS149B1, and PS167H2 cellular DNAfragments on Southern blots that hybridized with the approximately 800bp PS80JJ1 44.3 kDa toxin subgene probe under standard conditions StrainPS80JJ1 PS149B1 PS167H2 Restriction enzyme Approximate fragment size(kbp) EcoRI 6.4 5.7 2.6 1.3 2.8 0.6 HindIII 8.2 6.2 4.4 KpnI 7.8 10.011.5 PstI 12.0 9.2 9.2 8.2 XbaI 9.4 10.9 10.9 SacI 17.5 15.5 11.1 13.110.5 6.3

[0306] Each of the three strains exhibited unique RFLP patterns. Thehybridizing DNA fragments from PS149B1 or PS167H2 contain all or part oftoxin genes with sequence homology to the PS80JJ1 44.3 kDa toxin. TABLE7 Restriction fragment length polymorphisms of PS80JJ1, PS149B1, andPS167H2 cellular DNA fragments on Southern blots that hybridized withthe PS80JJ1 14 kDa toxin oligonucleotide probe under standard conditionsStrain PS80JJ1 PS149B1 PS167H2 Restriction enzyme Approximate fragmentsize (kbp) EcoRI 5.6 2.7 2.7 HindIII 7.1 6.0 4.7 XbaI 8.4 11.2 11.2

[0307] Each of the three strains exhibited unique RFLP patterns. Thehybridizing DNA fragments from PS149B1 or PS167H2 contain all or part oftoxin genes with sequence homology to the PS80JJ1 14 kDa toxin gene.

[0308] Portions of the toxin genes in PS149B1 or PS167H2 were amplifiedby PCR using forward and reverse oligonucleotide primer pairs designedbased on the PS80JJ1 44.3 kDa toxin gene sequence. For PS149B1, thefollowing primer pair was used: Forward: 5′-ATG YTW GAT ACW AAT AAA GTWTAT GAA AT-3′ (SEQ ID NO:8) Reverse: 5′-GGA TTA TCT ATC TCT GAG TGT TCTTG-3′ (SEQ ID NO:9)

[0309] For PS167H2, the same primer pair was used. These PCR-derivedfragments were sequenced using the ABI373 automated sequencing systemand associated software. The partial gene and peptide sequences obtainedare shown in SEQ ID NO:12-15. These sequences contain portions of thenucleotide coding sequences and peptide sequences for novel cornrootworm-active toxins present in B.t. strains PS149B1 or PS167H2.

Example 11 Molecular Cloning and DNA Sequence Analysis of Novelδ-Endotoxin Genes from Bacillus thuringiensis Strains PS149B1 andPS167H2

[0310] Total cellular DNA was extracted from strains PS149B1 and PS167H2as described for PS80JJ1. Gene libraries of size-fractionated Sau3Apartial restriction fragments were constructed in Lambda-Gem11 for eachrespective strain as previously described. Recombinant phage werepackaged and plated on E. coli KW251 cells. Plaques were screened byhybridization with the oligonucleotide probe specific for the 44 kDatoxin gene. Hybridizing phage were plaque-purified and used to infectliquid cultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

[0311] For PS167H2, Southern blot analysis revealed that one of therecombinant phage isolates contained an approximately 4.0 to 4.4 kbpHindIII band that hybridized to the PS80JJ1 44 kDa toxin gene 5′oligonucleotide probe (SEQ ID NO:8). This DNA restriction fragment wassubcloned by standard methods into pBluescript S/K (Stratgene, SanDiego, Calif.) for sequence analysis. The fragment was also subclonedinto the high copy number shuttle vector, pHT370 (Arantes, O., D.Lereclus [1991] Gene 108:115-119) for expression analyses in Bacillusthuringiensis (see below). The resultant recombinant, high copy numberbifunctional plasmid was designated pMYC2427.

[0312] The PS167H2 toxin genes encoded by pMYC2427 were sequenced usingthe ABI automated sequencing system and associated software. Thesequence of the entire genetic locus containing both open reading framesand flanking nucleotide sequences is shown in SEQ ID NO:34. Thetermination codon of the 14 kDa toxin gene is 107 base pairs upstream(5′) from the initiation codon of the 44 kDa toxin gene. The PS167H2 14kDa toxin coding sequence (SEQ ID NO:35), the 44 kDa toxin codingsequence (SEQ ID NO:37), and the respective deduced amino acidsequences, SEQ ID NO:36 and SEQ ID NO:38, are novel compared to otherknown toxin genes encoding pesticidal proteins. The toxin genes arearranged in a similar manner to, and have some homology with, thePS80JJ1 14 and 44 kDa toxins.

[0313] A subculture of E. coli NM522 containing plasmid pMYC2427 wasdeposited in the permanent collection of the Patent Culture Collection(NRRL), Regional Research Center, 1815 North University Street, Peoria,Ill. 61604 USA on 26 Mar. 1997. The accession number is NRRL B-21672.

[0314] For PS149B1, Southern blot analysis using the PS80JJ1 44 kDaoligonucleotide 5′ probe (SEQ ID NO:8) demonstrated hybridization of anapproximately 5.9 kbp ClaI DNA fragment. Complete ClaI digests ofPS149B1 genomic DNA were size fractionated on agarose gels and clonedinto pHTBlueII. The fragment was also subcloned into the high copynumber shuttle vector, pHT370 (Arantes, O., D. Lereclus [1991] Gene108:115-119) for expression analyses in Bacillus thuringiensis (seebelow). The resultant recombinant, high copy number bifunctional plasmidwas designated pMYC2429.

[0315] The PS149B1 toxin genes encoded by pMYC2429 were sequenced usingthe ABI automated sequencing system and associated software. Thesequence of the entire genetic locus containing both open reading framesand flanking nucleotide sequences is shown in SEQ ID NO:39. Thetermination codon of the 14 kDa toxin gene is 108 base pairs upstream(5′) from the initiation codon of the 44 kDa toxin gene. The PS149B1 14kDa toxin coding sequence (SEQ ID NO:40), the 44 kDa toxin codingsequence (SEQ ID NO:42), and the respective deduced amino acidsequences, SEQ ID NO:41 and SEQ ID NO:43, are novel compared to otherknown toxin genes encoding pesticidal proteins. The toxin genes arearranged in a similar manner as, and have some homology with, thePS80JJ1 and PS167H214 and 44 kDa toxins. Together, these three toxinoperons comprise a new family of pesticidal toxins.

[0316] A subculture of E. coli NM522 containing plasmid pMYC2429 wasdeposited in the permanent collection of the Patent Culture Collection(NRRL), Regional Research Center, 1815 North University Street, Peoria,Ill. 61604 USA on 26 Mar. 1997. The accession number is NRRL B-21673.

Example 12 PCR Amplification for Identification and Cloning Novel CornRootworm-Active Toxin

[0317] The DNA and peptide sequences of the three novel approximately 45kDa corn rootworm-active toxins from PS80JJ1, PS149B1, and PS167H2 (SEQID NOS.12-15) were aligned with the Genetics Computer Group sequenceanalysis program Pileup using a gap weight of 3.00 and a gap lengthweight of 0.10. The sequence alignments were used to identify conservedpeptide sequences to which oligonucleotide primers were designed thatwere likely to hybridize to genes encoding members of this novel toxinfamily. Such primers can be used in PCR to amplify diagnostic DNAfragments for these and related toxin genes. Numerous primer designs tovarious sequences are possible, four of which are described here toprovide an example. These peptide sequences are:Asp-Ile-Asp-Asp-Tyr-Asn-Leu (SEQ ID NO:16) Trp-Phe-Leu-Phe-Pro-Ile-Asp(SEQ ID NO:17) Gln-Ile-Lys-Thr-Thr-Pro-Tyr-Tyr (SEQ ID NO:18)Tyr-Glu-Trp-Gly-Thr-Glu. (SEQ ID NO:19)

[0318] The corresponding nucleotide sequences are:5′-GATATWGATGAYTAYAAYTTR-3′ (SEQ ID NO:20) 5′-TGGTTTTTRTTTCCWATWGAY-3′(SEQ ID NO:21) 5′-CAAATHAAAACWACWCCATATTAT-3′ (SEQ ID NO:22)5′-TAYGARTGGGGHACAGAA-3′. (SEQ ID NO:23)

[0319] Forward primers for polymerase amplification in thermocyclereactions were designed based on the nucleotide sequences of SEQ IDNOs:20 and 21.

[0320] Reverse primers were designed based on the reverse complement ofSEQ ID NOs:22 and 23: 5′-ATAATATGGWGTWGTTTTDATTTG-3′ (SEQ ID NO:24)5′-TTCTGTDCCCCAYTCRTA-3′. (SEQ ID NO:25)

[0321] These primers can be used in combination to amplify DNA fragmentsof the following sizes (Table 8) that identify genes encoding novel cornrootworm toxins. TABLE 8 Predicted sizes of diagnostic DNA fragments(base pairs) amplifiable with primers specific for novel cornrootworm-active toxins Primer pair (SEQ ID NO.) DNA fragment size (bp)20 + 24 495 20 + 25 594 21 + 24 471 21 + 25 580

[0322] Similarly, entire genes encoding novel corn rootworm-activetoxins can be isolated by polymerase amplification in thermocyclereactions using primers designed based on DNA sequences flanking theopen reading frames. For the PS80JJ1 44.3 kDa toxin, one such primerpair was designed, synthesized, and used to amplify a diagnostic 1613 bpDNA fragment that included the entire toxin coding sequence. Theseprimers are: Forward: 5′-CTCAAAGCGGATCAGGAG-3′ (SEQ ID NO:26) Reverse:5′-GCGTATTCGGATATGCTTGG-3′. (SEQ ID NO:27)

[0323] For PCR amplification of the PS80JJ1 14 kDa toxin, theoligonucleotide coding for the N-terminal peptide sequence (SEQ IDNO:29) can be used in combination with various reverse oligonucleotideprimers based on the sequences in the PS80JJ1 toxin gene locus. One suchreverse primer has the following sequence:

[0324] 5′CATGAGATTTATCTCCTGATCCGC 3′ (SEQ ID NO:33).

[0325] When used in standard PCR reactions, this primer pair amplified adiagnostic 1390 bp DNA fragment that includes the entire 14 kDa toxincoding sequence and some 3′ flanking sequences corresponding to the 121base intergenic spacer and a portion of the 44.3 kDa toxin gene. Whenused in combination with the 14 kDa forward primer, PCR will generate adiagnostic 322 base pair DNA fragment.

Example 13 Clone Dose-Response Bioassays

[0326] The PS80J11 toxin operon was subcloned from pMYC2421 into pHT370for direct comparison of bioactivity with the recombinant toxins clonedfrom PS149B1 and PS167H2. The resultant recombinant, high copy numberbifunctional plasmid was designated pMYC2426.

[0327] A subculture of E. coli NM522 containing plasmid pMYC2426 wasdeposited in the permanent collection of the Patent Culture Collection(NRRL), Regional Research Center, 1815 North University Street, Peoria,Ill. 61604 USA on 26 Mar. 1997. The accession number is NRRL B-21671.

[0328] To test expression of the PS80JJ1, PS149B1 and PS167H2 toxingenes in B.t., pMYC2426, pMYC2427 and pMYC2429 were separatelytransformed into the acrystalliferous (Cry-) B.t. host, CryB (A.Aronson, Purdue University, West Lafayette, Ind.), by electroporation.The recombinant strains were designated MR543 (CryB [pMYC2426]), MR544(CryB [pMYC2427]) and MR546 (CryB [pMYC2429]), respectively. Expressionof both the approximately 14 and 44 kDa toxins was demonstrated bySDS-PAGE analysis for each recombinant strain.

[0329] Toxin crystal preparations from the recombinant strains wereassayed against western corn rootworm. Their diet was amended withsorbic acid and SIGMA pen-strep-ampho-B. The material was top-loaded ata rate of 50 μl of suspension per cm² diet surface area. Bioassays wererun with neonate Western corn rootworm larvae for 4 days atapproximately 25° C. Percentage mortality and top-load LC₅₀ estimatesfor the clones (pellets) are set forth in Table 9. A dH₂O controlyielded 7% mortality. TABLE 9 Percentage mortality at given proteinconcentration (μg/cm²) Sample 50 μg/cm² 5 μg/cm² 0.5 μg/cm² MR543 pellet44% 19%  9% MR544 pellet 72% 32% 21% MR546 pellet 52% 32% 21%

[0330] The amounts of 14 kDa and 44.3 kDa proteins present in thecrystal preparations were estimated by densitometry and used tocalculate specific activity expressed as LC₅₀. LC₅₀ estimates for theclones (pellets) are set forth in Table 10 (WCRW top load bioassay ofB.t. clones). TABLE 10 WCRW Top Load Bioassay of B.t. Clones B.t. B.t.Parental LC₅₀ Clone Strain (μg/cm²)* 95% CL Slope MR543 PS80JJ1 37 17-366*  0.79 MR544 PS167H2 10 6-14 1.6 MR546 PS149B1  8 4-12 1.5 N/ACryB cell blank 4% N/A N/A N/A Water blank 4% N/A N/A

Example 14 Mutational Analysis of the 14 and 44 kDa Polypeptides in thePS80JJ1 binary Toxin Operon

[0331] Binary toxin genes of the subject invention are, in theirwild-type state, typically arranged in an operon wherein the 14 kDaprotein gene is transcribed first, followed by that of the 45 kDaprotein gene. These genes are separated by a relatively short,non-coding region. Representative ORFs are shown in SEQ ID NO:30, SEQ IDNO:34, and SEQ ID NO:39.

[0332] In order to investigate the contribution of the individual 14 and44.3 kDa crystal proteins to corn rootworm activity, each gene in thePS80JJ1 operon was mutated in separate experiments to abolish expressionof one of the proteins. The intact gene was then expressed in B.t. andrecombinant proteins were tested for activity against corn rootworm.

[0333] First, the 44.3 kDa gene encoded on pMYC2421 was mutated bytruncation at the EcoRI site at base position 387 of the open readingframe. This truncation and subsequent ligation with vector sequencesresulted in an open reading frame encoding an approximately 24 kDahypothetical fusion protein. The resulting operon encoding the intact 14kDa gene and the truncated 45 kDa gene was subcloned into the high copynumber shuttle vector, pHT370 (Arantes, O., D. Lereclus [1991] Gene108:115-119) for expression analyses in Bacillus thuringiensis. Theresulting plasmid, pMYC2424 was transformed into the acrystalliferous(Cry-) B.t. host, CryB (A. Aronson, Purdue University, West Lafayette,Ind.), by electroporation. The resulting recombinant strain wasdesignated MR541. Only the 14 kDa PS80JJ1 protein was detectable bySDSPAGE analysis of sporulated cultures of MR541. Mortality was notobserved for preparations of MR541 expressing only the 14 kDa PS80JJ1protein in top-load bioassays against corn rootworm.

[0334] Next, the 14 kDa gene encoded on pMYC2421 was mutated byinsertion of an oligonucleotide linker containing termination codons inall possible reading frames at the NruI site at base position 11 of theopen reading frame. The sequence of this linker is 5′TGAGTAACTAGATCTATTCAATTA 3′. The linker introduces a BglII site forconfirmation of insertion by BglII restriction digestion. Plasmid clonescontaining the mutagenic linker were identified with BglII and sequencedfor verification. The operon insert encoding the 14 kDa nonsensemutations was subcloned into pHT370, resulting in plasmid pMYC2425. Thisplasmid was transformed into CryB by electroporation to yield therecombinant B.t. strain MR542. Only the 44.3 kDa PS80JJ 1 protein wasexpressed in sporulated cultures of MR542 as shown by SDSPAGE analysis.Mortality against corn rootworm was not observed for preparations ofMR542 expressing only the 44.3 kDa PS80JJ1 protein.

Example 15 Single Gene Heterologous Expression, Purification andBioassay of the 14 and 44.3 kDa Polypeptides from PS149B1 in PseudomonasFluorescens

[0335] The 14 kDa and 44.3 kDa polypeptide genes from PS149B1 wereseparately engineered into plasmid vectors by standard DNA cloningmethods, and transformed into Psuedomonas flourescens. The recombinantPseudomonas fluorescens strain expressing only the PS149B1 14 kDa genewas designated MR1253. The recombinant Pseudomonas fluorescens strainexpressing only the PS149B1 44.3 kDa gene was designated MR1256.

[0336] MR1253 and MR1256 each individually expressing one of the twobinary proteins were grown in 1 L fermentation tanks. A portion of eachculture was then pelleted by centrifugation, lysed with lysozyme, andtreated with DNAse I to obtain semi-pure protein inclusions. Theseinclusions were then solubilized in 50 mM Sodium Citrate (pH 3.3) bygentle rocking at 4° C. for 1 hour. The 14 kDa protein dissolved readilyin this buffer whereas the 44.3 kDa protein was partially soluble. Thesolubilized fractions were then centrifuged at 15,000×g for 20 minutes;and the supernatants were retained.

[0337] The 14 kDa protein was further purified through ion-exchangechromatography. The solubilized 14 kDa protein was bound to a Econo-Scolumn and eluted with a Sodium Chloride 0-1M gradient.

[0338] The chromatographically pure MR1253 (14 kDa protein) and theSodium Citrate (pH3.3) solubilized preparation of MR1256 (45 kDaprotein) were then tested for activity on corn rootworm individually ortogether at a molar ratio of 1 to 10 (45 kDa protein to 14 kDa protein).Observed mortality for each of the proteins alone was not abovebackground levels (of the water/control sample) but 87% mortalityresulted when they were combined in the above ratio (see Table 11).TABLE 11 Molar ratio load ug 45 kD/ ug 14 kD/ Total ug CRW (45 kD to 14kD) volume well well protein Mortality 0 to 1 100 ul 0 260 260 13 1 to 0200 ul 260 0 260 9 1 to 10 100 ul 65 195 260 87 water 100 ul 0 0 0 11

Example 16 Identification of Additional Novel 14 kDa and 44.3 kDa ToxinGenes by Hybridization of Total B.t. Genomic DNA and by RFLP

[0339] Total genomic DNA from each isolate was prepared using the QiagenDNEasy 96 well tissue kit. DNA in 96-well plates was denatured prior toblotting by adding 10 ul of each DNA sample and 10 ul of 4 M NaOH to 80ul sterile distilled water. Samples were incubated at 70° C. for onehour after which 100 ul of 20×SSC was added to each well. PS149B1 totalgenomic DNA was included with each set of 94 samples as a positivehybridization control, and cryB- total genomic DNA was included witheach set of 94 samples as a negative hybridization control. Each set of96 samples was applied to Magnacharge nylon membranes using two 48 wellslot blot manifolds (Hoefer Scientific), followed by two washes with10×SSC. Membranes were baked at 80° C. for one hour and kept dry untilused. Membranes were prehybridized and hybridized in standard formamidesolution (50% formamide, 5×SSPE, 5× Denhardt's solution, 2% SDS, 100ug/ml single stranded DNA) at 42° C. Membranes were washed under twoconditions: 2×SSC/0.1% SDS at 42° C. (low stringency) and 0.2×SSC/0.1%SDS at 65° C. (moderate to high stringency). Membranes were probed withan approximately 1.3 kilobase pair PCR fragment of the PS149B1 44.3 kDagene amplified from pMYC2429 using forward primer SEQ ID NO:8 and areverse primer with the sequence 5′ GTAGAAGCAGAACAAGAAGGTATT 3′ (SEQ IDNO:46). The probe was radioactively labeled using the Prime-it II kit(Stratagene) and 32-P-dCTP, purified on Sephadex columns, denatured at94° C. and added to fresh hybridization solution. Strains containinggenes with homology to the PS149B1 probe were identified by exposingmembranes to X-ray film.

[0340] The following strains were identified by positive hybridizationreactions: PS184M2, PS185GG, PS187G1, PS187Y2, PS201G, PS201HH2,PS242K10, PS69Q, KB54A1-6, KR136, KR589, PS185L12, PS185W3, PS185Z11,PS186L9, PS187L14, PS186FF, PS131W2, PS147U2, PS158T3, PS158X10,PS185FF, PS187F3, PS198H3, PS201H2, PS201L3, PS203G2, PS203J1, PS204C3,PS204G4, PS204I11, PS204J7, PS210B, PS213E8, PS223L2, PS224F2, PS236B6,PS246P42, PS247C16, KR200, KR331, KR625, KR707, KR959, KR1209, KR1369,KB2C-4, KB10H-5, KB456, KB42C17-13, KB45A43-3, KB54A33-1, KB58A10⁻³,KB59A54-4, KB59A54-5, KB53B7-8, KB53B7-2, KB60F5-7, KB60F5-11,KB59A58-4, KB60F5-15, KB61A18-1, KB65A15-2, KB65A15-3, KB65A15-7,KB65A15-8, KB65A15-12, KB65A14-1, KB3F-3, T25, KB53A71-6, KB65A11-2,KB68B57-1, KB63A5-3, and KB71A118-6.

[0341] Further identification and classification of novel toxin genes inpreparations of total genomic DNA was performed using the ³²P-labeledprobes and hybridization conditions described above in this Example.Total genomic DNA was prepared as above or with Qiagen Genomic-Tip 20/Gand Genomic DNA Buffer Set according to protocol for Gram positivebacteria (Qiagen Inc.; Valencia, Calif.) was used in southern analysis.For Southern blots, approximately 1-2 μg of total genomic DNA from eachstrain identified by slot blot analysis was digested with Dral and NdeIenzymes, electrophoresed on a 0.8% agarose gel, and immobilized on asupported nylon membrane using standard methods (Maniatis et al.). Afterhybridization, membranes were washed under low stringency (2×SSC/0.1%SDS at 42° C.) and exposed to film. DNA fragment sizes were estimatedusing BioRad Chemidoc system software. Restriction fragment lengthpolymorphisms were used to (arbitrarily) classify genes encoding the 44kDa toxin. These classifications are set forth in Table 12. TABLE 12RFLP Class (45 & 14 kD) Isolate Strain Name A 149B1  A′ KR331, KR1209,KR1369 B 167H2, 242K10 C 184M2, 201G, 201HH2 D 185GG, 187Y2, 185FF1,187F3 E 187G1 F 80JJ1, 186FF, 246P42 G 69Q H KB54A1-6 I KR136 J KR589 K185L12, 185W3, 185Z11, 186L9, 187L14 L 147U2, 210B, KB10H-5, KB58A10-3,KB59A54-4, KB59A54-5, KB59A58-4, KB65A14-1 M 158T3, 158X10 N 201H2,201L3, 203G2, 203J1, 204C3, 204G4, 204I11, 204J7, 236B6 P 223L2, 224F2P′ 247C16, KB45A43-3, KB53B7-8, KB53B7-2, KB61A18-1, KB3F-3, KB53A71-6,KB65A11-2, KB68B57-1, KB63A5-3, KB71A118-6 Q 213E8, KB60F5-11, KB60F5-15R KR959 S KB2C-4, KB46, KB42C17-13 T KB54A33-1, KB60F5-7 U T25 VKB65A15-2, KB65A15-3, KB65A15-7, KB65A15-8, KB65A15-12

Example 17 DNA sequencing of Additional Binary Toxin Genes

[0342] Degenerate oligonucleotides were designed to amplify all or partof the 14 and 44.3 kDa genes from B.t. strains identified byhybridization with the 149B1 PCR product described above. Theoligonucleotides were designed to conserved sequence blocks identifiedby alignment of the 14 kDa or 44.3 kDa genes from PS149B1, PS167H2 andPS80JJ1. Forward primers for both genes were designed to begin at theATG initiation codon. Reserve primers were designed as close to the 3′end of each respective gene as possible.

[0343] The primers designed to amplify the 14 kDa gene are as follows:149DEG1 (forward): 5′-ATG TCA GCW CGY GAA GTW CAY ATT G-3′ (SEQ IDNO:47) 149DEG2 (reverse): 5′-GTY TGA ATH GTA TAH GTH ACA TG-3′ (SEQ IDNO:48)

[0344] These primers amplify a product of approximately 340 base pairs.

[0345] The primers designed to amplify the 44.3 kDa gene are as follows:149DEG3 (forward): 5′-ATG TTA GAT ACW AAT AAA RTW TAT G-3′ (SEQ IDNO:49) 149DEG4 (reverse): 5′-GTW ATT TCT TCW ACT TCT TCA TAH GAA G-3′(SEQ ID NO:50)

[0346] These primers amplify a product of approximately 1,100 basepairs.

[0347] The PCR conditions used to amplify gene products are as follows:

[0348] 95° C., 1 min., one cycle

[0349] 95° C., 1 min.

[0350] 50° C., 2 min., this set repeated 35 cycles

[0351] 72° C., 2 min.

[0352] 72° C., 10 min., one cycle

[0353] PCR products were fractionated on 1% agarose gels and purifiedfrom the gel matrix using the Qiaexll kit (Qiagen). The resultingpurified fragments were ligated into the pCR-TOPO cloning vector usingthe TOPO TA cloning kit (Invitrogen). After ligation, one half of theligation reaction was transformed into XL10 Gold ultracompetant cells(Stratagene). Transformants were then screened by PCR with vectorprimers 1212 and 1233. Clones containing inserts were grown on theLB/carbenicillin medium for preparation of plasmids using the Qiagenplasmid DNA miniprep kit (Qiagen). Cloned PCR-derived fragments werethen sequenced using Applied Biosystems automated sequencing systems andassociated software. Sequences of additional novel binary toxin genesand polypeptides related to the holotype 14 and 44.3 kDa toxins fromPS80JJ1 and PS149B1 are listed as SEQ ID NOS. 51-126. The section above,entitled “BriefDescription of the Sequences,” provides a furtherexplanation of these sequences.

[0354] The 14 kDa-type toxins and genes from three additional B.t.strains, PS137A, PS201 V2 and PS207C3, were also sequenced using theabove procedures (with any differences noted below). PCR using the149DEG 1 (forward) and 149DEG2 (reverse) primers was performed. Theseprimers amplify a product of approximately 340 base pairs. The PCR wasperformed with the following conditions:

[0355] 1. 95° C., 3 min.

[0356] 2. 94° C., 1 min.

[0357] 3. 42° C., 2 min.

[0358] 4. 72° C., 3 min.+5 sec./cycle

[0359] 5. Steps 2 through 4 repeated 29 times

[0360] PCR products were gel purified using the QiaQuick gel extractionkit (Qiagen), the purified fragment was ligated into the pCR-TOPOcloning vector using the TOPO-TA kit (Invitrogen), and subsequentlytransformed into XL10-Gold Ultracompetent E. coli cells (Strategene).Preparation of transformant DNA is described above. Sequences of the 14kDa toxin gene for each of the three new strains were obtained as perabove. The nucleotide and polypeptide sequences are provided in theattached Sequence Listing as follows: PS137A (SEQ ID NOs:149 and 150),PS201V2 (SEQ ID NOs:151 and 152), and PS207C3 (SEQ ID NOs:153 and 154).

Example 18 PS149B1 Toxin Transgenes and Plant Transformation

[0361] Separate synthetic transgenes optimized for maize codon usagewere designed for both the 14 and 44.3 kDa toxin components. Thesynthetic versions were designed to modify the guanine and cytosinecodon bias to a level more typical for plant DNA. Preferredplant-optimized transgenes are described in SEQ ID NOs: 127-128. Thepromoter region used for expression ofboth transgenes was the Zea maysubiquitin promoter plus Z. mays exon 1 and Z. mays intron 1(Christensen, A. H. et al. (1992) Plant Mol. Biol. 18:675-689). Thetranscriptional terminator used for both transgenes was the potatoproteinase inhibitor II (PinII) terminator (An, G. et al. 1989 PlantCell 1:115-22).

[0362] Phosphinothricin acetyltransferase (PAT) was used as theselectable marker for plant transformation. The phosphinothricinacetyltransferase gene (pat) was isolated from the bacteriumStreptomyces viridochromogenes (Eckes P. et al., 1989). The PAT proteinacetylates phosphinothricin, or its precursor demethylphosphinothricin,conferring tolerance to a chemically synthesized phosphinothricin suchas the herbicide glufosinate-ammonium. Acetylation convertsphosphinothricin to an inactive form that is no longer toxic to cornplants. Glufosinate ammonium is a broad spectrum, non-systemic,non-selective herbicide. Regenerating corn tissue or individual cornplants tolerant to glufosinate ammonium herbicide can be readilyidentified through incorporation of PAT into regeneration medium or byspray application of the herbicide to leaves.

[0363] The synthetic version of the pat gene was produced in order tomodify the guanine and cytosine codon bias to a level more typical forplant DNA. The promoter for the pat gene is the CaMV promoter of the 35Stranscript from cauliflower mosiac virus (Pietrzak et al., 1986). Thetranscriptional terminator is the CaMV 35 S terminator.

[0364] For transformation of maize tissue, a linear portion of DNA,containing both the PS149B 114 and 44.3 kDa andpat selectable markercoding sequences, and the regulatory components necessary forexpression, was excised from a complete plasmid. This linear portion ofDNA, termed an insert, was used in the transformation process.

[0365] Maize plants containing PS149B1 14 kDa and 44.3 kDa transgeneswere obtained by microprojectile bombardment using the Biolistics®OPDS-100He particle gun manufactured by Bio-Rad, essentially as describedby Klein et al. (1987). Immature embryos isolated from corn earsharvested approximately 15 days after pollination were cultured oncallus initiation medium for three to eight days. On the day oftransformation, microscopic tungsten particles were coated with purifiedDNA and accelerated into the cultured embryos, where the insert DNA wasincorporated into the cell chromosome. Six days after bombardment,bombarded embryos were transferred to callus initiation mediumcontaining glufosinate (Bialaphos) as the selection agent. Healthy,resistant callus tissue was obtained and repeatedly transferred to freshselection medium for approximately 12 weeks. Plants were regenerated andtransferred to the greenhouse. A total of 436 regenerated plants wereobtained. Leaf samples were taken for molecular analysis to verify thepresence of the transgenes by PCR and to confirm expression of theforeign protein by ELISA. Plants were then subjected to a whole plantbioassay using western corn rootworm. Positive plants were crossed withinbred lines to obtain seed from the initial transformed plants. Theseplants were found to be resistant to damage by corn rootworm in bothgreenhouse and field trials.

Example 19 Further Bioassays

[0366] Protein preparations from the strains identified on Example 16were assayed for activity against western corn rootworm using the basictop load assay methods, as described in Example 13. TABLE 13 Strain LC₅₀(ug/cm2) 95% Cl KB45A43-3 9.48  6.58-15.27 213‘E’8 10.24  7.50-19.87KR707 11.17  8.27-22.54^(#) 185GG 11.53  7.51-16.81 187Y2 13.82 11.08-17.67 149B1 14.77  4.91-27.34 69Q 27.52 117.28-114.77^(#) 167H231.38  19.35-47.60 KB54A33-10 32.62  24.76-83.85 185Z11 34.47 NDKB60F5-7 34.67  19.15-124.29 242K10 34.73  21.08-58.25 201G 34.90 13.20-355.18^(#) 204J7 38.57  29.83-48.82 KB60F5-15 38.62 15.00-2.59E03 80JJ1 41.96  27.35-139.43 203J1 43.85  23.18-69.51 KR58947.28  29.83-230.71^(#) 201HH2 49.94  23.83-351.77 KB60F5-11 51.84 19.38-1313.75^(#) 158X10 52.25  43.13-77.84^(#) KB58A10-3 53.77 ND201L3 55.01  41.01-78.96 158T3 58.07  39.59-211.13 184M2 60.54 26.57-411.88 204G4 69.09  52.32-93.83 KB59A58-4 70.35  48.90-144.90201H2 71.11  52.40-130.35 203G2 81.93  57.13-226.33 KB59A54-4 82.03 38.50-1.63E03 204I11 88.41  62.48-173.07 236B6 89.33  64.16-158.96KR1369 93.25  71.97-205.04^(#) KB63A5-3 94.52  51.56-542.46 204C3 125.45 85.26-427.67^(#) KR1209 128.14  91.57-294.56 185W3 130.61 ND KR625160.36 ND 210B 201.26  48.51-0.14E+06^(#) KB10H-5 214.25  87.97-8.22E+03KB68B57-1 264.30  48.51-8.95E+04^(#) 223L2 3.81E+02 ND KR136 7.83E+02 —T25 1.30E+03 ND KB61A18-1 2.58E+03 ND 147U2 3.67E+03 ND KR200 2.14E+05ND KB59A54-5 3.32E+05 ND KB3F-3 4.07E+05 ND 187G1(bs) 3.50E+07 ND MR55920%** n/a KB42C17-13 26%** n/a 224F2 33%** n/a KR959 41%** n/a KB2C-442%** n/a 198H3 46%** n/a KR331 47%** n/a KB46 55%** n/a KB71A118-671%** n/a KB53B7-2 84%** n/a 187Y2 ND n/a 185L12 ND ND 186L9 ND n/aKB54A1-6 ND n/a 187L14 ND n/a 187G1(b) nt nt 187G1(s) nt nt

Example 20 Molecular Cloning Expression, and DNA Sequence Analysis of aNovel Binary Endotoxin Gene from Bacillus thuringiensis Strain PS201L3.

[0367] Genomic DNA from PS201 L3 was prepared from cells grown in shakeflask culture using a Qiagen Genomic-tip 500/G kit and Genomic DNABuffer Set according to the protocol for Gram positive bacteria (QiagenInc.; Valencia, Calif.). A gene library was constructed from PS201L3 DNApartially digested with Sau3AI. Partial restriction digests werefractionated by agarose gel electrophoresis. DNA fragments 9.3 to 23 kbpin size were

[0368] excised from the gel, electroeluted from the gel slice, purifiedon an Elutip-D ion exchange column (Schleicher and Schuell, Keene,N.H.), and recovered by ethanol precipitation. The Sau3AI inserts wereligated into BamHI-digested LambdaGem-11 (Promega, Madison, Wis.).Recombinant phage were packaged using Gigapack III XL Packaging Extract(Stratagene, La Jolla, Calif.) and plated on E. coli KW251 cells.Plaques were lifted onto Nytran Nylon Transfer Membranes(Schleicher&Schuell, Keene, N.H.) and probed with a ³²P-dCTP labeledgene probe for binary toxin coding sequences. This gene probe was anapproximately 1.0 kb PCR product amplified using genomic PS201L3 DNAtemplate and oligonucleotides “15kfor1” and “45krev6.”

[0369] The sequences of the oligonucleotides used for PCR and sequencingfollow: 15kfor1 (SEQ ID NO:131) ATGTCAGCTCGCGAAGTACAC 45krev6 (SEQ IDNO:132) GTCCATCCCATTAATTGAGGAG

[0370] The membranes were hybridized with the probe overnight at 65° C.and then washed three times with 1×SSPE and 0.1% SDS. Thirteen plaqueswere identified by autoradiography. These plaques were subsequentlypicked and soaked overnight in 1 mL SM Buffer+10uL CHCl₃. Phage wereplated for confluent lysis on KW251 host cells; 6 confluent plates weresoaked in SM and used for large-scale phage DNA preparations. Thepurified phage DNA was digested with various enzymes and run on 0.7%agarose gels. The gels were transferred to Nytran Membranes by Southernblotting and probed with the same PCR-amplified DNA fragment as above.An approximately 6.0 kb hybridizing XbaI band was identified andsubcloned into pHT370, an E. coli/Bacillus thuringiensis shuttle vector(Arantes, O., D. Lereclus [1991] Gene 108:115-119) to generate pMYC2476.XL10 Gold Ultracompetent E. coli cells (Stratagene) transformed withpMYC2476 were designated MR1506. PMYC2476 was subsequently transformedinto acrytalliferous CryB cells by electroporation and selection onDM3+erythromycin (20ug/mL) plates at 30° C. Recombinant CryB[pMYC2476]was designated MR561.

[0371] A subculture of MR1506 was deposited in the permanent collectionof the Patent Culture Collection (NRRL), Regional Research Center, 1815North University Street, Peoria,

[0372] Illinois 61604 USA on Jun. 1, 2000. The accession number isB-30298.

[0373] B.t. strain MR561 was examined for expression of the PS201L3binary toxin proteins by immunoblotting. Cells were grown in liquidNYS-CAA medium+erythromycin (10 ug/ml) overnight at 30° C. The culturewas then pelleted by centrifugation and a portion of the cell pellet wasresuspended and run on SDS-PAGE gels. Both 14 kDa and 44 kDa proteinswere apparent by Western Blot analysis when probed with antibodiesspecific for either the PS149B1 14 kDa or 44 kDa toxins, respectively.

[0374] DNA sequencing of the toxin genes encoded on pMYC2476 wasperformed using an

[0375] ABI377 automated sequencer. The DNA sequence for PS201L3 14 kDagene is shown in SEQ ID NO:133. The deduced peptide sequence for PS201L314 kDa toxin is shown in SEQ ID NO:134. The DNA sequence for PS201 L3 44kDa gene is shown in SEQ ID NO:135. The deduced peptide sequence forPS201 L3 44 kDa toxin is shown in SEQ ID NO:136.

[0376] The following table shows sequence similarity and identityofbinary genes and proteins from 201 L3 and 149B 1. The program BESTFIT(part of the GCG software package) was used for these comparisons.BESTFIT uses the local homology algorithm of Smith and Waterman(Advances in Applied Mathematics 2: 482-489 (1981)). TABLE 14 201L3 vs149B1 % similarity % identity 14 kDa nucleotide seq — 71.1 14 kDapeptide seq 63.9 54.1 45 kDa nucleotide seq — 76.1 45 kDa peptide seq70.9 62.7

Example 21 Molecular Cloning and DNA Sequence Analysis of Novel6-Endotoxin Genes from Bacillus thuringiensis Strains PS187G1, PS201HH2and KR1369

[0377] Total cellular DNA was prepared from Bacillus thuringensisstrains PS187G1, PS201HH2 and KR1369 grown to an optical density of0.5-1.0 at 600 nm visible light in Luria Bertani (LB) broth. DNA wasextracted using the Qiagen Genomic-tip 500/G kit and Genomic DNA BufferSet according to the protocol for Gram positive bacteria (Qiagen Inc.;Valencia, Calif.). PS187G1, PS201HH2 and KR1369 cosmid libraries wereconstructed in the SuperCosl vector (Stratragene) using inserts ofPS187G1, PS201HH2 and KR1369 total cellular DNA, respectively, partiallydigested with Nde II. XL1-Blue MR cells (Stratagene) were transfectedwith packaged cosmids to obtain clones resistant to carbenicillin andkanamycin. For each strain, 576 cosmid colonies were grown in 96-wellblocks in 1 ml LB+carbenicillin (100 ug/ml)+kanamycin (50 ug/ml) at 37°C. for 18 hours and replica plated onto nylon filters for screening byhybridization.

[0378] PCR amplicon containing approximately 1000 bp of the PS187G1,PS201HH2 or KR1369 14 kDa and 44 kDa toxin operon was amplified fromPS187G1, PS201HH2 or KR1369 genomic DNA using primers designed toamplify binary homologs: 15kfor1: 5′-ATG TCA GCT CGC GAA GTA CAC-3′ (SEQID NO:131) 45krev6: 5′-GTC CAT CCC ATT AAT TGA GGA G-3′ (SEQ ID NO:132)

[0379] The DNA fragment was gel purified using QiaQuick extraction(Qiagen). The probe was radiolabeled with ³²P-dCTP using the Prime-It IIkit (Stratgene) and used in aqueous hybridization solution (6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA) with the colonylift filters at 65° C. for 16 hours. The colony lift filters werebriefly washed 1×in 0.5×SSC/0.1% SDS at room temperature followed by twoadditional washes for 10 minutes at65° C. in 0.5×SSC/0.1% SDS. Thefilters were then exposed to X-ray film for 20 minutes (PS187G1 andPS201HH2) or for 1 hour (KR1369). One cosmid clone that hybridizedstrongly to the probe was selected for further analysis for each strain.These cosmid clones were confirmed to contain the approximately 1000 bp14 kDa and 44 kDa toxin gene target by PCR amplification with theprimers listed above. The cosmid clone of PS187G1 was designated aspMYC3106; recombinant E. coli XLl-Blue MR cells containing pMYC3106 aredesignated MR1508. The cosmid clone of PS201HH2 was designated aspMYC3107; recombinant E. coli XL1-Blue MR cells containing pMYC3107 aredesignated MR1509. The cosmid clone of KR1369 was designated aspMYC3108; recombinant E. coli XL1-Blue MR cells containing pMYC3108 aredesignated MR1510. Subcultures of MR1509 and MR1510 were deposited inthe permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Aug. 8, 2000. The accession numbers are NRRL B-30330 andNRRL-B 30331, respectively.

[0380] The PS187G1, PS201HH2 and KR1369 14 kDa and 44 kDa toxin genesencoded by pMYC3106, pMYC3107 and pMYC3108, respectively, were sequencedusing the AB1377 automated sequencing system and associated software.

[0381] The PS187G1 14 kDa and 44 kDanucleotide and deduced polypeptidesequences are shown as SEQ ID NOs: 137-140. Both the 14 kDa and 44 kDatoxin gene sequences are complete open reading frames. The PS187G1 14kDa toxin open reading frame nucleotide sequence, the 44 kDa toxin openreading frame nucleotide sequence, and the respective deduced amino acidsequences are novel compared to other toxin genes encoding pesticidalproteins.

[0382] The PS201HH2 14 kDa and 44 kDa nucleotide and deduced polypeptidesequences are shown as SEQ ID NOs:141-144. The 14 kDa toxin genesequence is the complete open reading frame. The 44 kDa toxin genesequence is a partial sequence of the gene. The PS201HH2 14 kDa toxinopen reading frame nucleotide sequence, the 44 kDa toxin partial openreading frame nucleotide sequence, and the respective deduced amino acidsequences are novel compared to other toxin genes encoding pesticidalproteins.

[0383] The KR1369 14 kDa and 44 kDa nucleotide and deduced polypeptidesequences are shown as SEQ ID NOs:145-148. Both the 14 kDa and 44 kDatoxin gene sequences are complete open reading frames. The KR1369 14 kDatoxin open reading frame nucleotide sequence, the 44 kDa toxin openreading frame nucleotide sequence, and the respective deduced amino acidsequences are novel compared to other toxin genes encoding pesticidalproteins.

Example 22 Construction and Expression of a Hybrid Gene FusionContaining the PS149B1 14 kDa and 44 kDa Binary Toxin Genes

[0384] Oligonucleotide primers were designed to the 5′ and 3′ ends ofboth the 14 kDa and 44 kDa genes from PS149B1. These oligonucleotideswere designed to create a gene fusion by SOE-PCR (“Gene Splicing ByOverlap Extension: Tailor-made Genes Using PCR,” Biotechniques8:528-535, May 1990). The two genes were fused together in the reverseorder found in the native binary toxin operon (i.e. 44 kDa gene first,followed by the 14 kDa gene.)

[0385] The sequences of the olignucleotides used for SOE-PCR were thefollowing: F1new: AAATATTATTTTATGTCAGCACGTGAAGTACACATTG (SEQ ID NO:155)R1new: tctctGGTACCttaTTAtgatttatgcccatatcgtgagg (SEQ ID NO:156) F2new:agagaACTAGTaaaaaggagataaccATGttagatactaataaag (SEQ ID NO:157) R2new:CGTGCTGACATAAAATAATATTTTTTTAATTTTTTTAGTGTACTTT (SEQ ID NO:158)

[0386] Oligo “F1new” was designed to direct amplification from the 5′end of the 14 kDa gene and hybridize to the 3′ end of the 44 kDa gene.Oligo “R1new” was designed to direct amplification from the 3′ end ofthe 14 kDa gene. This primer was designed with two stop codons in orderto ensure termination of translation. It was also designed with a KpnIsite for directional cloning into a plasmid expression vector forPseudomonas fluorescens. Oligo “F2new” was designed to directamplification from the 5′ end of the 44 kDa gene. It also includes aribosome binding sequence and a SpeI cloning site. Oligo “R2new” wasdesigned to direct amplification from the 3′ end of the 44 kDa gene andhybridize to the 5′ end of the 14 kDa gene.

[0387] The two genes were first independently amplified from PS149B;genomic DNA; the 14 kDa gene using “F1new” and “R1 new,” and the 44 kDagene using “F2new” and “R2new.” The products were then combined in onePCR tube and amplified togetherusing “R1new” and “F2new.” At this point,HerculaseTM Enhanced Polymerase Blend (Stratagene, La Jolla, Calif.) wasused at a 48° C. annealing temperature to amplify a ˜1.5 kb DNA fragmentcontaining the gene fusion. This DNA fragment was subsequently digestedusing KpnI and SpeI, fractionated on agarose gels, and purified byelectroelution. The plasmid vector was also digested with KpnI and SpeI,fractionated on agarose gels, purified by electroelution and treatedwith phosphatase. The vector and insert were then ligated togetherovernight at 14° C. Ligated DNA fragments were transformed into MB214P.f. cells by electroporation and selection overnight on LB+tetracycline (30 ug/mL) plates. Strains containing the gene fusion wereidentified by diagnostic PCR and sequenced for verification ofsuccessful gene splicing. One representative strain containing thecloned gene fusion was designated MR1607; the recombinant plasmid wasdesignated pMYC2475.

[0388] A subculture of MR1607 was deposited in the permanent collectionof the Patent Culture Collection (NRRL), Regional Research Center, 1815North University Street, Peoria, Ill. 61604 USA on Aug. 8, 2000. Theaccession number is NRRL B-30332. MR1607 was grown and proteinproduction was verified by SDS-PAGE and immunoblotting. A protein bandat ˜58 kDa representing the 44 kDa+14 kDa fusion product was identifiedwhen western blots were probed with antibodies specific to either the 14kDa toxin or the 44 kDa toxin.

[0389] The sequence of the 58 kDa fusion protein is provided in SEQ IDNO:159. The DNA sequence for the gene fusion is provided in SEQ IDNO:160.

Example 23 Binary Homologue Mixing Study Growth of Homologue Strains.

[0390] Four strains were selected, one from each major binary toxinfamily—149B1, 80JJ1, 201L3, and 167H2. In order to reduce time spentpurifying individual toxin proteins, the following Pseudomonasfluorescens (P.f.) clones were grown instead: MR1253 (14 kDa of 149B1)and MR1256 (44 kDa of 149B1). Similarly, B.t. clones MR541 (expressing14 kDa of 80JJ1), and MR542 (44 kDa of 80JJ1) were used. B.t. strainswere grown as described in Example 1. Pellets were washed 3× with waterand stored at −20° C. until needed. Pf strains were grown in 10 Lbatches in Biolafitte fermenters using standard procedures. Pellets werestored at −80° C. until needed.

[0391] Extraction & Purification of Toxins.

[0392] Purification of 167H2, MR541, MR542, 201L3. Extractions of cellpellets were done using 100 mM sodium citrate buffer at pH's rangingfrom 3.0 to 5.5. In a typical extraction, pellets were extracted with abuffer volume {fraction (1/10)} to ⅓ of the original culture volume.Pellets were suspended in the buffer and placed on a rocking platform at4° C. for periods of time ranging from 2.5 hours to overnight. Theextracts were centrifuged and supernatants were retained. This procedurewas repeated with each strain until at least approximately 10 mg of eachprotein were obtained. SDS-PAGE confirmed the presence/absence ofprotein toxins in the extracts through use of the NuPAGE Bis/Tris gelsystem (Invitrogen). Samples were prepared according to themanufacturer's instructions and were loaded onto 4-12% gels and theelectrophoretograms were developed with MES running buffer. Theexception to this procedure was the sample prep of all 201L3 samples.These samples were prepared by diluting ½× with BioRad's Laemmli samplebuffer and heating at 95° C. for 4 minutes. Protein quantitation wasdone by laser scanning gel densitometry with BSA as a standard(Molecular Dynamics Personal Densitometer SI). Extracts were clarifiedby filtration through a 0.2 μm membrane filter and stored at 4° C.

[0393] Purification of MR1253 & MR1256. The recombinant proteins MR1253and MR 1256, corresponding to the 14 and 44 kDa proteins of 149B1respectively, were prepared as solubilized inclusions. Inclusion bodieswere prepared using standard procedures. The inclusion bodies weresolubilized in 1 mM EDTA, 50 mM sodium citrate, pH 3.5.

[0394] Purification ofindividual toxins, 167H2 & 201L3. All extractsknown to contain either the 14, the 44 kDa, or both were combined. Thiscombined extract was dialyzed against 100 mM sodium citrate, 150 mMNaCl, pH 4. Dialysis tubing was from Pierce (Snakeskin 10k MWCO).Samples were usually dialyzed for approximately 6 hours and then againovernight in fresh buffer.

[0395] Extracts were then concentrated with either Centriprep 10 orCentricon Plus-20 (Biomax-5, 5000 NMWL) centrifugal filter devices(Millipore), quantitated for both the 14 kDa and 44 kDa proteins, andsubjected to gel filtration chromatography.

[0396] In preparation for chromatography, all samples and buffers werefiltered through a 0.2 μm filter and degassed. Samples were then appliedto a HiPrep 26/60 Sephacryl S-100 gel filtration column which had beenequilibrated with two bed volumes of the separation buffer, 100 mMsodium citrate, 150 mM NaCl, pH 4.0. Sample volumes ranged from 5-10 ml.An AKTA purifier 100 FPLC system (Amersham Pharmacia) controlled theruns. Chromatography was done at ambient temperature. Buffer flowthrough the column during the run was maintained at 0.7 ml/min. Proteinswere detected by monitoring UV absorbance at 280 nm. Fractions werecollected and stored at 4° C. Fractions containing either the 14 or 44kDa protein were pooled and checked for purity by SDS-PAGE as describedabove.

[0397] For 167H2 samples, two large peaks were detected and were wellseparated from each other at the baseline. SDS-PAGE of fractions showedeach peak represented one of the protein toxins.

[0398] In the 201 L3 sample, three well defined peaks and one shoulderpeak were detected. SDS-PAGE revealed that the first peak represented a100 kDa protein plus an 80 kDa protein. The second peak represented the44 kDa protein, while the shoulder peak was a 40 kDa protein. The thirdpeak was the 14 kDa protein. Fractions with the 44 kDa from both sampleswere combined as were all fractions containing the 14 kDa.

[0399] The 149B1 proteins had been obtained individually from Pf clonesMR1253 and MR1256 and, therefore, further purification was notnecessary. Similarly, the 80JJ1 recombinants, MR541 and MR542 yieldedthe individual 14 and 44 kDa proteins thereby obviating furtherpurification.

[0400] Sample Preparation for wCRW LC₅₀ Bioassay.

[0401] Dialysis. Samples of individual binary toxin proteins weredialyzed against 6 L of 20 mM sodium citrate, pH 4.0. The first dialysisproceeded for several hours, the samples were transferred to freshbuffer and alowed to dialyze overnight. Finally, the samples weretransferred to fresh buffer and dialyzed several more hours. Sources ofthe protein samples were either the pooled gel-filtration fractions(167H2, 201L3), pellet extracts (MR541, MR542), or inclusion pelletextracts (MR1253, MR1256). All samples were filtered through 0.2 ummembranes to sterilize.

[0402] Concentration. Samples were concentrated with Centricon Plus-20(Biomax-5, 5000 NMWL) centrifugal filter devices (Millipore).

[0403] Quantitation. Samples were quantitated for protein as above. Tomeet the requirements of the LC₅₀ bioassay, a minimum of 6.3 mg of eachtoxin protein were needed at a concentration range of 0.316-1.36 mg/mlfor the various combinations. If necessary, samples were concentrated asabove, or were diluted with buffer (20 mM sodium citrate, pH 4.0) andrequantitated.

[0404] Mixing of binaries/LC₅₀ bioassay. For each of the four strains,the 14 kDa was combined with an amount the 44 kDa of each strain to givea 1/1 mass ratio. The top dose was 50 ug/cm² for the mixtures, with theexception of mixtures with the 14 kDa protein of 203J1. Top doses ofmixtures with this protein were only 44 ug/cm². For controls, eachprotein was submitted individually, as was the extract buffer, 20 mMsodium citrate, pH 4.0. Native combinations were also tested (i.e. 14kDa+44 kDa of 149B1). All toxin combinations and buffer controls wereevaluated three times by bioassay against Western corn rootworm, whileindividual toxins were tested once.

[0405] The results are reported below in Table 15 (LC₅₀ Results forToxin Combinations) and Table 16 (Comparison of Potencies of Strains to149B1). TABLE 15 Toxin combination Top load, ul/well LC₅₀ (ug/cm2) 80JJ114 + 80JJ1 44 96 28 (19-44 C.I.) 167H2 44 159 >Top dose 201L3 44 172 Nodose response 149B1 44 78 No dose response 167H2 14 + 167H2 44 161 19(13-27 C.I.) 80JJ1 44 97 No dose response 201L3 44 174 14 (10-22 C.I.)149B1 44 80 No dose response 201L3 14 + 201L3 44 193 No dose response80JJ1 44 116 No dose response 167H2 44 180 No dose response 149B1 44 99No dose response 149B1 14 + 149B1 44 45 10 (7-15 C.I.) 80JJ1 44 63 11(8-16 C.I.) 167H2 44 126  8 (6-11 C.I.) 201L3 44 139 18 (13-27 C.I.)

[0406] TABLE 16 Comparison of potencies of strains to 149B1 Toxincombination Relative potency 149B1 14 + 149B1 44 To which all others arecompared 149B1 14 + 80JJ1 44 0.9 149B1 14 + 167H2 44 1.3 149B1 14 +201L3 44 0.5 80JJ1 14 + 80JJ1 44 0.4 167H2 14 + 167H2 44 0.5 167H2 14 +201L3 44 0.7

[0407] The results are also displayed graphically in FIG. 3.

[0408] Native combinations were highly active against Western cornrootworm, except for 201 L3. However, the 44 kDa of 201L3 was activewhen combined with either the 14 kDa of 167H2 or 149B1. Other activecombinations were the 149B1 14 kDa with either 80JJ1 or 167H2 44 kDa,with the latter appearing to be more active than the native 149B1mixture. No dose response was noted for either the individual proteins,or the buffer and water controls.

Example 24 Control of Southern Corn Rootworm with PS149B1 14-kDa Protein

[0409] A powder containing approximately 50% (wt/wt) of a 14-kDa8-endotoxin, originally discovered in Bacillus thuringiensis strainPS149B 1, was isolated from recombinant Pseudomonas fluorescens strain(MR1253). This powder was evaluated for insecticidal activity using thefollowing procedure.

[0410] Artificial insect diet (R. I. Rose and J. M. McCabe (1973),“Laboratory rearing techniques for rearing corn rootworm,” J. Econ.Entomol. 66 (2): 398-400) was dispensed at ˜0.5 mL/well into 128-wellbioassay trays (C-D International, Pitman, N.J.) to produce a surfacearea of ˜1.5 cm2. Buffer (10-mM potassium phosphate, pH 7.5) suspensionsof the 14-kDa protein powder were applied to the surface of theartificial insect diet at 50 μL/well, and the diet surface was allowedto dry. Buffer controls were also included in each test. A singleneonate southern corn rootworm, Diabrotica undecimpunctata howardi, wasplaced in each well, and the wells were sealed with lids that wereprovided with the trays. The bioassays were held for 6 days at 28° C.,after which time, the live larvae were weighed as a group for eachtreatment. Percent growth inhibition was calculated by subtracting theweight of live insects from each treatment from the weight of live,control insects, and then dividing by the control weight. This resultwas multiplied by 100 to convert the number to a percent. Growthinhibition was calculated for each of 5 tests that each contained 16insects per treatment, and the growth inhibition was averaged acrosstests.

[0411] Results demonstrated that the 14-kDa protein inhibited growth ofsouthern corn rootworms in a concentration-dependent manner. Table 17shows southern corn rootworm growth inhibition with PS149B1 14-kDaprotein. TABLE 17 Concentration % Growth Treatment in μg ai/cm²Inhibition 14-kDa Protein 1 32 14-kDa Protein 3 55 14-kDa Protein 9 78

Example 25 Control of European Corn Borer and Corn Earworm with PS149B1Binary Toxin

[0412] A powder containing 54% of a 14-kDa δ-endotoxin, and anotherpowder containing 37% of a 44-kDa δ-endotoxin, both originallydiscovered in Bacillus thuringiensis strain PS149B1, were isolated fromrecombinant Pseudomonas fluorescens strains MR1253 and MR1256,respectively. Mixtures of these powders were evaluated for insecticidalactivity using the following procedure.

[0413] Artificial insect diet (R. I. Rose and J. M. McCabe (1973),“Laboratory rearing techniques for rearing corn rootworm,” J. Econ.Entomol. 66 (2): 398-400) was dispensed at ˜0.5 mL/well into 128-wellbioassay trays (C-D International, Pitman, N.J.) to produce a surfacearea of 1.5 cm2. Buffer (10-mM potassium phosphate, pH 7.5) suspensionsof the protein powders were mixed, and were then applied to the surfaceof the artificial insect diet at 50 μL/well. The diet surface wasallowed to dry. Buffer controls were also included in each test. Asingle neonate larvae was placed in each well, and the wells were sealedwith lids that were provided with the trays. Tests were conducted withEuropean corn borer, Ostrinia nubilalis, and corn earworm, Helicoverpazea (both are lepidopterans). The bioassays were held for 6 days at 28°C., after which time, the live larvae were weighed as a group for eachtreatment. Percent growth inhibition was calculated by subtracting theweight of live insects in each treatment from the weight of live,control insects, and then dividing by the control weight. This resultwas multiplied by 100 to convert the number to a percent. Growthinhibition was calculated for each of 4 tests that each contained 14 to16 insects per treatment, and the growth inhibition was averaged acrosstests.

[0414] Results demonstrated that the 14-kDa protein inhibited growth ofEuropean corn borers and corn earworms in a concentration-dependentmanner. Table 18 shows corn earworm (CEW) and European corn borer (ECB)growth inhibition with PS149B1 protein mixtures. TABLE 18 14-kDaprotein + % Growth 44-kDa Protein Inhibition Concentration in μg ai/cm2CEW ECB 3.7 + 11  42 59 11 + 33 57 77  33 + 100 61 89

Example 26 Further Characterization of the 45 kDa Proteins and PrimerDesign for Identifying Additional Polynucleotides and Proteins

[0415] The subject invention includes not only the specificallyexemplified sequences. Portions of the subject genes and toxins can beused to identify other related genes and toxins. Thus, the subjectinvention includes polynucleotides that encode proteins or polypeptidescomprising at least ten contiguous amino acids, for example, of any ofthe binary-type proteins or polypeptides included in the attachedsequence listing and described herein. Other embodiments includepolynucleotides that encode, for example, at least 20, 30, 40, 50, 60,70, 80, 90, and 100 contiguous amino acids of a protein exemplifiedherein; these numbers also apply similarly to contiguous nucleotides ofan exemplified polynucleotide. The proteins encoded by suchpolynucleotides are included in the subject invention. Likewise,polynucleotides comprising contiguous nucleotides (that code forproteins or polypeptides comprising peptides of these approximate sizes)are included in the subject invention.

[0416] While still very different, the “closest” toxins to those of thesubject invention are believed to be the 51 and 42 kDa mosquitocidalproteins of Bacillus sphaericus. Attached as FIGS. 4 and 5 are proteinalignments and nucleotide sequences alignments of the 51 and 42 kDasphaericus toxins and genes and the 45 kDa 149B1 toxin and gene.

[0417] Two blocks of sequences are highlighted in the nucleotidealignment to which primers could be made. An exemplary PCR primer pairis included below, and in 5′-3′ orientation (45 kD3′rc is shown as thecomplement). These primers have been successfully used to identifyadditional members of the 45 kDa binary family. Fully redundantsequences and a prophetic pair are also included below. 45kD5′: (SEQ IDNO:161) GAT RAT RAT CAA TAT ATT ATT AC. 45kD3′rc: (SEQ ID NO:162) CAAGGT ART AAT GTC CAT CC.

[0418] The sequences would be useful as both the sequence written and asthe reverse complement (03 and 04 are complementary to 45 kD3′rc, theexemplified reverse primer). 45kD5′01: (SEQ ID NO:163) GAT GATGrTmrAKwwATTATTrC A. 45kD5′02: (SEQ ID NO:164) GAT GATGrTmrAT ATATTATTrC A.45kD3′03: (SEQ ID NO:165) GGAwG krCdyTwdTm CCwTGTAT. 45kD3′04: (SEQ IDNO:166) GGAwG kACryTAdTA CCTTGTAT.

[0419] Regarding the manner in which the sphaericus toxins wereidentified, a BLAST (Altschul et al. (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs,”Nucleic Acids Res. 25:3389-3402) database search using the 149B1 45 kDaprotein found matches to the 42 kDa B. sphaericus crystal inclusionprotein (expectation score 3*10⁻¹⁴) and the 51 kDa B. sphaericus crystalinclusion protein (expectation score 3*10⁻⁹).

[0420] An alignment of the 45 kDa 149B 1 peptide sequence to the 42 kDaB. sphaericus crystal inclusion protein results in an alignment having26% identity over 325 residues. The alignment score is 27.2 sd above themean score of 100 randomized alignments. A similar analysis of the 45kDa 149B1 peptide sequence to the 42 kDa B. sphaericus crystal inclusionprotein results in an alignment having 29% identity over 229 residues.The alignment score is 23.4 sd above the mean score of 100 randomizedalignments. Alignment scores >10 sd above the mean of random alignmentshave been considered significant (Lipman, D. J. and Pearson, W. R.(1985), “Rapid and sensitive similarity searches,” Science227:1435-1441; Doolittle, R. F. (1987), Of URFs and ORFs: a primer onhow to analyze derived amino acid sequences, University Science Books,Mill Valley, Calif.).

[0421] For reference, the structurally similar Cry1 Aa, Cry2Aa andCry3Aa protein sequences were compared in the same way. Cry2Aa vs.Cry1Aa and Cry2Aa vs. Cry3Aa share 29% and 27% identity over 214 and 213residues, respectively, with alignment scores 32.2 sd and 29.5 sd abovethe mean score of 100 randomized alignments. An alignment of the 149B145 kDa protein sequence and the Cry2Aa protein sequence resulted in analignment score within 1 sd of the mean of 100 randomized alignments.

[0422] The following comparisons are also noted: TABLE 19 % AverageComparison Quality Length Ratio Gaps % Similarity Identity Quality*ps149b1-45.pep x 189 325 0.612 12 35.135 26.351 39.4 ± 5.5 s07712ps149b1-45.pep x 161 229 0.742 9 36.019 28.910 39.3 ± 5.2 07711cry2aa1.pep x 182 214 0.888 6 37.688 28.643 43.5 ± 4.3 cry1aa1.pepcry3aa1.pep x 187 213 0.926 6 40.500 27.000 42.3 ± 4.9 cry2aa1.pepps149b1-45.pep x 40 28 1.429 0 42.857 35.714 41.6 ± 5.6 cry2aa1.pep

[0423] For further comparison purposes, and for further primer design,the following references are noted:

[0424] Oei et al. (1992), “Binding of purified Bacillus sphaericusbinary toxin and its deletion derivatives to Culex quinquefasciatus gut:elucidation of functional binding domains,” Journal of GeneralMicrobiology 138(7): 1515-26.

[0425] For the 51 kDa: 35-448 is active; 45-448 is not; 4-396 is active;4-392 is not.

[0426] For the 42 kDa: 18-370 is active, 35-370 is not; 4-358 is active;4-349 is not.

[0427] The work was done with GST fusions purified and cleaved withthrombin. All truncations were assayed with ==of other intact subunit.All deletions had some loss of activity. P51 deltaC56 binds, but doesn'tinternalize 42. P51delta N45 doesn't bind. Only 42 kDa+51 kDa areinternalized. Both N-terminal and C-terminal non-toxic 42 kDa proteinsfailed to bind the 51 kDa protein or 51 kDa-receptor complex.

[0428] Davidson et al. (1990), “Interaction of the Bacillus sphaericusmosquito larvicidal proteins,” Can. J. Microbiol. 36(12):870-8.N-termini of SDS-PAGE purified proteins obtained from B. sphaericus. S29and N31 of 51 kDa and S9 of 42 kDa in 68-74 kDa complexes (unreduced).S9 and S29 of 51 and N31 of 42 from 51 kDa band (unreduced). In reducedgels the 45 kDa band had S29 and N31 of the 51 kDa and the 39 kDa bandcontained S9 of the 42 kDa protein.

[0429] Baumann et al. (1988), “Sequence analysis of the mosquitocidaltoxin genes encoding 51.4- and 41.9-kilodalton proteins from Bacillussphaericus 2362 and 2297,” J. Bacteriol. 17:2045-2050. N-termini of 41.9kDa at D5 from B. sphaericus protease and I11 from chymotrypsin;C-terminus following R349 with trypsin. Regions of enhanced similaritywere identified that correspond to many of those above. Similar sequenceblocks A through D between the 51 and 42 kDa proteins.

[0430] In summary, the sphaericus toxins discussed above are not meantto be included in the scope of the subject invention (in fact, they arespecifically excluded). In that regard, divergent contiguous sequences,as exemplified in the alignments (FIGS. 4 and 5) discussed above, can beused as primers to identify unique toxins that are suggested but notspecifically exemplified herein. However, the conserved contiguoussequences, as shown in the alignments, can also be used according to thesubject invention to identify further novel 15/45 kDa-type binary toxins(active against corn rootworm and other pests).

Example 27 Insertion and Expression of Toxin Genes In Plants

[0431] One aspect of the subject invention is the transformation ofplants with polynucleotides of the subject invention that expressproteins of the subject invention. The transformed plants are resistantto attack by the target pest.

[0432] The novel corn rootworm-active genes described here can beoptimized for expression in other organisms. For example, maizeoptimized gene sequences encoding the 14 and 44 kDa PS80JJ1 toxins aredisclosed in SEQ ID NO:44 and SEQ ID NO:45, respectively.

[0433] Genes encoding pesticidal toxins, as disclosed herein, can beinserted into plant cells using a variety of techniques which are wellknown in the art. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher plants. The vectors comprise, forexample, pBR322, pUC series, M13 mp series, pACYC184, etc. Accordingly,the sequence encoding the B.t. toxin can be inserted into the vector ata suitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or R1 plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted.

[0434] The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120 516; Hoekema(1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci.4:1-46; and An et al. (1985) EMBO J. 4:277-287.

[0435] Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

[0436] A large number of techniques are available for inserting DNA intoa plant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens orAgrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

[0437] The transformed cells grow inside the plants in the usual manner.They can form germ cells and transmit the transformed trait(s) toprogeny plants. Such plants can be grown in the normal manner andcrossed with plants that have the same transformed hereditary factors orother hereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

[0438] In a preferred embodiment of the subject invention, plants willbe transformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831, which is herebyincorporated by reference. Also, advantageously, plants encoding atruncated toxin will be used. The truncated toxin typically will encodeabout 55% to about 80% of the full length toxin. Methods for creatingsynthetic B.t. genes for use in plants are known in the art.

Example 28 Cloning of B.t. Genes Into Insect Viruses

[0439] A number of viruses are known to infect insects. These virusesinclude, for example, baculoviruses and entomopoxviruses. In oneembodiment of the subject invention, genes encoding the insecticidaltoxins, as described herein, can be placed within the genome of theinsect virus, thus enhancing the pathogenicity of the virus. Methods forconstructing insect viruses which comprise B.t. toxin genes are wellknown and readily practiced by those skilled in the art. Theseprocedures are described, for example, in Merryweather et al.(Merryweather, A. T., U. Weyer, M. P. G. Harris, M. Hirst, T. Booth, R.D. Possee (1990) J. Gen. Virol. 71:1535-1544) and Martens et al.(Martens, J. W. M., G. Honee, D. Zuidema, J. W. M. van Lent, B. Visser,J. M. Vlak (1990) Appl. Environmental Microbiol. 56(9):2764-2770).

[0440] All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification

[0441] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

1 166 1 5 PRT Bacillus thuringiensis 1 Met Leu Asp Thr Asn 1 5 2 25 PRTBacillus thuringiensis 2 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser AsnLeu Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu 20 25 3 24PRT Bacillus thuringiensis 3 Ser Ala Arg Glu Val His Ile Glu Ile Asn AsnThr Arg His Thr Leu 1 5 10 15 Gln Leu Glu Ala Lys Thr Lys Leu 20 4 25PRT Bacillus thuringiensis 4 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile SerAsn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu 20 255 50 PRT Bacillus thuringiensis MISC_FEATURE (35)..(35) Undeterminedamino acid 5 Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly HisThr 1 5 10 15 Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg TrpArg Thr 20 25 30 Ser Pro Xaa Asn Val Ala Asn Asp Gln Ile Lys Thr Phe ValAla Glu 35 40 45 Ser Asn 50 6 25 PRT Bacillus thuringiensis 6 Met LeuAsp Thr Asn Lys Ile Tyr Glu Ile Ser Asn Tyr Ala Asn Gly 1 5 10 15 LeuHis Ala Ala Thr Tyr Leu Ser Leu 20 25 7 25 PRT Bacillus thuringiensis 7Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 1 5 1015 Leu Gln Leu Glu Asp Lys Thr Lys Leu 20 25 8 29 DNA ArtificialSequence Oligonucleotide probe for gene encoding PS80JJ1 44.3 kDa toxin;forward primer for PS149B1 and PS167H2 8 atgntngata cnaataaagt ntatgaaat29 9 26 DNA Artificial Sequence Reverse primer for PS149B1 and PS167H2 9ggattatcta tctctgagtg ttcttg 26 10 1158 DNA Bacillus thuringiensis 10atgttagata ctaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatca 60acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120gatgattaca atttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatc tcttggaata 360gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720acaactatta ttaatacagt aggattgcaa attaatatag attcaggaat gaaatttgaa 780gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900aatccaacta atcaaccaat gaattctata ggacttctta tttatacttc tttagaatta 960tatcgatata acggtacaga aattaagata atggacatag aaacttcaga tcatgatact 1020tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattcg 1080tatgaagaag tagaagaaat aacaaaaata cctaagcata cacttataaa attgaaaaaa 1140cattatttta aaaaataa 1158 11 385 PRT Bacillus thuringiensis 11 Met LeuAsp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala Asn Gly 1 5 10 15 LeuTyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 MetSer Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 LeuPhe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 AsnAsn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Ile Asn Val Ser 65 70 75 80Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Phe Pro 115120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Glu Lys Leu Lys Asp His ProGlu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Asn Pro Lys Thr Thr ProGln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn AspSer Lys Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr TyrIle Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn Leu Ala Lys Gly Ser AsnVal Ser Leu Leu Pro His 210 215 220 Gln Lys Arg Ser Tyr Asp Tyr Glu TrpGly Thr Glu Lys Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr ValGly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Glu Val ProGlu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265 270 Thr Gln Leu Thr GluGlu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280 285 Ile Met Thr LysTyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Pro MetAsn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu Glu Leu 305 310 315 320 TyrArg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp Ile Glu Thr Ser 325 330 335Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Lys Glu Ala 340 345350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355360 365 Lys Ile Pro Lys His Thr Leu Ile Lys Leu Lys Lys His Tyr Phe Lys370 375 380 Lys 385 12 834 DNA Bacillus thuringiensis 12 ggactatatgcagcaactta tttaagttta gatgattcag gtgttagttt aatgaataaa 60 aatgatgatgatattgatga ttataactta aaatggtttt tatttcctat tgatgatgat 120 caatatattattacaagcta tgcagcaaat aattgtaaag tttggaatgt taataatgat 180 aaaataaatgtttcgactta ttcttcaaca aattcaatac aaaaatggca aataaaagct 240 aatggttcttcatatgtaat acaaagtgat aatggaaaag tcttaacagc aggaaccggt 300 caagctcttggattgatacg tttaactgat gaatcctcaa ataatcccaa tcaacaatgg 360 aatttaacttctgtacaaac aattcaactt ccacaaaaac ctataataga tacaaaatta 420 aaagattatcccaaatattc accaactgga aatatagata atggaacatc tcctcaatta 480 atgggatggacattagtacc ttgtattatg gtaaatgatc caaatataga taaaaatact 540 caaattaaaactactccata ttatatttta aaaaaatatc aatattggca acgagcagta 600 ggaagtaatgtagctttacg tccacatgaa aaaaaatcat atacttatga atggggcaca 660 gaaatagatcaaaaaacaac aattataaat acattaggat ttcaaatcaa tatagattca 720 ggaatgaaatttgatatacc agaagtaggt ggaggtacag atgaaataaa aacacaacta 780 aatgaagaattaaaaataga atatagtcat gaaactaaaa taatggaaaa atat 834 13 278 PRT Bacillusthuringiensis 13 Gly Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser GlyVal Ser 1 5 10 15 Leu Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr AsnLeu Lys Trp 20 25 30 Phe Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile ThrSer Tyr Ala 35 40 45 Ala Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp LysIle Asn Val 50 55 60 Ser Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp GlnIle Lys Ala 65 70 75 80 Asn Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn GlyLys Val Leu Thr 85 90 95 Ala Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg LeuThr Asp Glu Ser 100 105 110 Ser Asn Asn Pro Asn Gln Gln Trp Asn Leu ThrSer Val Gln Thr Ile 115 120 125 Gln Leu Pro Gln Lys Pro Ile Ile Asp ThrLys Leu Lys Asp Tyr Pro 130 135 140 Lys Tyr Ser Pro Thr Gly Asn Ile AspAsn Gly Thr Ser Pro Gln Leu 145 150 155 160 Met Gly Trp Thr Leu Val ProCys Ile Met Val Asn Asp Pro Asn Ile 165 170 175 Asp Lys Asn Thr Gln IleLys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys 180 185 190 Tyr Gln Tyr Trp GlnArg Ala Val Gly Ser Asn Val Ala Leu Arg Pro 195 200 205 His Glu Lys LysSer Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln 210 215 220 Lys Thr ThrIle Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser 225 230 235 240 GlyMet Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile 245 250 255Lys Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr 260 265270 Lys Ile Met Glu Lys Tyr 275 14 829 DNA Bacillus thuringiensis 14acatgcagca acttatttaa gtttagatga ttcaggtgtt agtttaatga ataaaaatga 60tgatgatatt gatgactata atttaaggtg gtttttattt cctattgatg ataatcaata 120tattattaca agctacgcag cgaataattg taaggtttgg aatgttaata atgataaaat 180aaatgtttca acttattctt caacaaactc gatacagaaa tggcaaataa aagctaatgc 240ttcttcgtat gtaatacaaa gtaataatgg gaaagttcta acagcaggaa ccggtcaatc 300tcttggatta atacgtttaa cggatgaatc accagataat cccaatcaac aatggaattt 360aactcctgta caaacaattc aactcccacc aaaacctaca atagatacaa agttaaaaga 420ttaccccaaa tattcacaaa ctggcaatat agacaaggga acacctcctc aattaatggg 480atggacatta ataccttgta ttatggtaaa tgatcccaat atagataaaa acactcaaat 540caaaactact ccatattata ttttaaaaaa atatcaatat tggcaacaag cagtaggaag 600taatgtagct ttacgtccgc atgaaaaaaa atcatatgct tatgagtggg gtacagaaat 660agatcaaaaa acaactatca ttaatacatt aggatttcag attaatatag attcgggaat 720gaaatttgat ataccagaag taggtggagg tacagatgaa ataaaaacac aattaaacga 780agaattaaaa atagaatata gccgtgaaac caaaataatg gaaaaatat 829 15 276 PRTBacillus thuringiensis 15 His Ala Ala Thr Tyr Leu Ser Leu Asp Asp SerGly Val Ser Leu Met 1 5 10 15 Asn Lys Asn Asp Asp Asp Ile Asp Asp TyrAsn Leu Arg Trp Phe Leu 20 25 30 Phe Pro Ile Asp Asp Asn Gln Tyr Ile IleThr Ser Tyr Ala Ala Asn 35 40 45 Asn Cys Lys Val Trp Asn Val Asn Asn AspLys Ile Asn Val Ser Thr 50 55 60 Tyr Ser Ser Thr Asn Ser Ile Gln Lys TrpGln Ile Lys Ala Asn Ala 65 70 75 80 Ser Ser Tyr Val Ile Gln Ser Asn AsnGly Lys Val Leu Thr Ala Gly 85 90 95 Thr Gly Gln Ser Leu Gly Leu Ile ArgLeu Thr Asp Glu Ser Pro Asp 100 105 110 Asn Pro Asn Gln Gln Trp Asn LeuThr Pro Val Gln Thr Ile Gln Leu 115 120 125 Pro Pro Lys Pro Thr Ile AspThr Lys Leu Lys Asp Tyr Pro Lys Tyr 130 135 140 Ser Gln Thr Gly Asn IleAsp Lys Gly Thr Pro Pro Gln Leu Met Gly 145 150 155 160 Trp Thr Leu IlePro Cys Ile Met Val Asn Asp Pro Asn Ile Asp Lys 165 170 175 Asn Thr GlnIle Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr Gln 180 185 190 Tyr TrpGln Gln Ala Val Gly Ser Asn Val Ala Leu Arg Pro His Glu 195 200 205 LysLys Ser Tyr Ala Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys Thr 210 215 220Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly Met 225 230235 240 Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys Thr245 250 255 Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser Arg Glu Thr LysIle 260 265 270 Met Glu Lys Tyr 275 16 7 PRT Artificial Sequence Peptidesequence used in primer design 16 Asp Ile Asp Asp Tyr Asn Leu 1 5 17 7PRT Artificial Sequence Peptide sequence used in primer design 17 TrpPhe Leu Phe Pro Ile Asp 1 5 18 8 PRT Artificial Sequence Peptidesequence used in primer design 18 Gln Ile Lys Thr Thr Pro Tyr Tyr 1 5 196 PRT Artificial Sequence Peptide sequence used in primer design 19 TyrGlu Trp Gly Thr Glu 1 5 20 21 DNA Artificial Sequence Nucleotidesequence corresponding to the peptide of SEQ ID NO16 20 gatatngatgantayaaytt n 21 21 21 DNA Artificial Sequence Nucleotide sequencecorresponding to the peptide of SEQ ID NO17 21 tggtttttnt ttccnatnga n21 22 24 DNA Artificial Sequence Nucleotide sequence corresponding tothe peptide of SEQ ID NO18 22 caaatnaaaa cnacnccata ttat 24 23 18 DNAArtificial Sequence Nucleotide sequence corresponding to the peptide ofSEQ ID NO19 23 tangantggg gnacagaa 18 24 24 DNA Artificial SequenceReverse primer based on the reverse complement of SEQ ID NO22 24ataatatggn gtngttttna tttg 24 25 18 DNA Artificial Sequence Reverseprimer based on the reverse complement of SEQ ID NO23 25 ttctgtnccccantcnta 18 26 18 DNA Artificial Sequence Forward primer based on thePS80JJ1 44.3 kDa toxin 26 ctcaaagcgg atcaggag 18 27 20 DNA ArtificialSequence Reverse primer based on the PS80JJ1 44.3 kDa toxin 27gcgtattcgg atatgcttgg 20 28 386 PRT Artificial Sequence Generic sequencerepresenting a new class of toxins 28 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Thr Tyr LeuSer Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Xaa Lys Xaa Asp Xaa AspIle Asp Asp Tyr Asn Leu Xaa Trp Phe 35 40 45 Leu Phe Pro Ile Asp Xaa XaaGln Tyr Ile Ile Thr Ser Tyr Xaa Ala 50 55 60 Asn Asn Cys Lys Val Trp AsnVal Xaa Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr AsnSer Xaa Gln Lys Trp Gln Ile Lys Ala Xaa 85 90 95 Xaa Ser Ser Tyr Xaa IleGln Ser Xaa Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Xaa Gly Gln XaaLeu Gly Xaa Xaa Arg Leu Thr Asp Glu Xaa Xaa 115 120 125 Xaa Asn Xaa AsnGln Gln Trp Asn Leu Thr Xaa Val Gln Thr Ile Gln 130 135 140 Leu Pro XaaLys Pro Xaa Ile Asp Xaa Lys Leu Lys Asp Xaa Pro Xaa 145 150 155 160 TyrSer Xaa Thr Gly Asn Ile Xaa Xaa Xaa Thr Xaa Pro Gln Leu Met 165 170 175Gly Trp Thr Leu Xaa Pro Cys Ile Met Val Asn Asp Xaa Xaa Ile Asp 180 185190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Xaa Lys Lys Tyr 195200 205 Xaa Tyr Trp Xaa Xaa Ala Xaa Gly Ser Asn Val Xaa Leu Xaa Pro His210 215 220 Xaa Lys Xaa Ser Tyr Xaa Tyr Glu Trp Gly Thr Glu Xaa Xaa GlnLys 225 230 235 240 Thr Thr Ile Ile Asn Thr Xaa Gly Xaa Gln Ile Asn IleAsp Ser Gly 245 250 255 Met Lys Phe Xaa Xaa Pro Glu Val Gly Gly Gly ThrXaa Xaa Ile Lys 260 265 270 Thr Gln Leu Xaa Glu Glu Leu Lys Xaa Glu TyrSer Xaa Glu Thr Lys 275 280 285 Ile Met Xaa Lys Tyr Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa 38529 28 DNA Artificial Sequence Oligonucleotide probe 29 gngaagtncatatngaaatn aataatac 28 30 2015 DNA Bacillus thuringiensis 30 attaattttatggaggttga tatttatgtc agctcgcgaa gtacacattg aaataaacaa 60 taaaacacgtcatacattac aattagagga taaaactaaa cttagcggcg gtagatggcg 120 aacatcacctacaaatgttg ctcgtgatac aattaaaaca tttgtagcag aatcacatgg 180 ttttatgacaggagtagaag gtattatata ttttagtgta aacggagacg cagaaattag 240 tttacattttgacaatcctt atataggttc taataaatgt gatggttctt ctgataaacc 300 tgaatatgaagttattactc aaagcggatc aggagataaa tctcatgtga catatactat 360 tcagacagtatctttacgat tataaggaaa atttataaaa actgtatttt ttactaaaat 420 accaaaaaatacatatttat tttttggtat tttctaatat gaaatatgaa ttataaaaat 480 attaataaaaaaggtgataa aaattatgtt agatactaat aaagtttatg aaataagcaa 540 tcttgctaatggattatata catcaactta tttaagtctt gatgattcag gtgttagttt 600 aatgagtaaaaaggatgaag atattgatga ttacaattta aaatggtttt tatttcctat 660 tgataataatcaatatatta ttacaagcta tggagctaat aattgtaaag tttggaatgt 720 taaaaatgataaaataaatg tttcaactta ttcttcaaca aactctgtac aaaaatggca 780 aataaaagctaaagattctt catatataat acaaagtgat aatggaaagg tcttaacagc 840 aggagtaggtcaatctcttg gaatagtacg cctaactgat gaatttccag agaattctaa 900 ccaacaatggaatttaactc ctgtacaaac aattcaactc ccacaaaaac ctaaaataga 960 tgaaaaattaaaagatcatc ctgaatattc agaaaccgga aatataaatc ctaaaacaac 1020 tcctcaattaatgggatgga cattagtacc ttgtattatg gtaaatgatt caaaaataga 1080 taaaaacactcaaattaaaa ctactccata ttatattttt aaaaaatata aatactggaa 1140 tctagcaaaaggaagtaatg tatctttact tccacatcaa aaaagatcat atgattatga 1200 atggggtacagaaaaaaatc aaaaaacaac tattattaat acagtaggat tgcaaattaa 1260 tatagattcaggaatgaaat ttgaagtacc agaagtagga ggaggtacag aagacataaa 1320 aacacaattaactgaagaat taaaagttga atatagcact gaaaccaaaa taatgacgaa 1380 atatcaagaacactcagaga tagataatcc aactaatcaa ccaatgaatt ctataggact 1440 tcttatttatacttctttag aattatatcg atataacggt acagaaatta agataatgga 1500 catagaaacttcagatcatg atacttacac tcttacttct tatccaaatc ataaagaagc 1560 attattacttctcacaaacc attcgtatga agaagtagaa gaaataacaa aaatacctaa 1620 gcatacacttataaaattga aaaaacatta ttttaaaaaa taaaaaacat aatatataaa 1680 tgactgattaatatctctcg aaaaggttct ggtgcaaaaa tagtgggata tgaaaaaagc 1740 aaaagattcctaacggaatg gaacattagg ctgttaaatc aaaaagttta ttgataaaat 1800 atatctgcctttggacagac ttctcccctt ggagagtttg tccttttttg accatatgca 1860 tagcttctattccggcaatc atttttgtag ctgtttgcaa ggattttaat ccaagcatat 1920 ccgaatacgctttttgataa ccgatgtctt gttcaatgat attgtttaat attttcacac 1980 gaattggctactgtgcggta tcctgtctcc tttat 2015 31 360 DNA Bacillus thuringiensis 31atgtcagctc gcgaagtaca cattgaaata aacaataaaa cacgtcatac attacaatta 60gaggataaaa ctaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120gatacaatta aaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180atatatttta gtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240ggttctaata aatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300ggatcaggag ataaatctca tgtgacatat actattcaga cagtatcttt acgattataa 360 32119 PRT Bacillus thuringiensis 32 Met Ser Ala Arg Glu Val His Ile GluIle Asn Asn Lys Thr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys ThrLys Leu Ser Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala ArgAsp Thr Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr GlyVal Glu Gly Ile Ile Tyr Phe Ser 50 55 60 Val Asn Gly Asp Ala Glu Ile SerLeu His Phe Asp Asn Pro Tyr Ile 65 70 75 80 Gly Ser Asn Lys Cys Asp GlySer Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95 Ile Thr Gln Ser Gly Ser GlyAsp Lys Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Val Ser Leu ArgLeu 115 33 24 DNA Artificial Sequence Reverse oligonucleotide primer 33catgagattt atctcctgat ccgc 24 34 2230 DNA Bacillus thuringiensis 34actatgacaa tgattatgac tgctgatgaa ttagctttat caataccagg atattctaaa 60ccatcaaata taacaggaga taaaagtaaa catacattat ttactaatat aattggagat 120attcaaataa aagatcaagc aacatttggg gttgtttttg atccccctct taatcgtatt 180tcaggggctg aagaatcaag taagtttatt gatgtatatt atccttctga agatagtaac 240cttaaatatt atcaatttat aaaagtagca attgattttg atattaatga agattttatt 300aattttaata atcatgacaa tatagggata tttaattttg ttacacgaaa ttttttatta 360aataatgaaa atgattaata aaaaatttaa tttgtataat atgtttattt tttgaaaatt 420gaatgcatat attaatcgag tatgtgtaat aaattttaat tttatggagg ttgatattta 480tgtcagcacg tgaagtacac attgatgtaa ataataagac aggtcataca ttacaattag 540aagataaaac aaaacttgat ggtggtagat ggcgaacatc acctacaaat gttgctaatg 600atcaaattaa aacatttgta gcagaatcac atggttttat gacaggtaca gaaggtacta 660tatattatag tataaatgga gaagcagaaa ttagtttata ttttgacaat ccttattcag 720gttctaataa atatgatggg cattccaata aaaatcaata tgaagttatt acccaaggag 780gatcaggaaa tcaatctcat gttacgtata ctattcaaac tgtatcttca cgatatggga 840ataattcata aaaaaatatt tttttttacg aaaataccaa aaaaattttt ttggtatttt 900ctaatataat tcataaatat tttaataata aaattataag aaaaggtgat aaatattatg 960ttagatacta ataaaattta tgaaataagt aattatgcta atggattaca tgcagcaact 1020tatttaagtt tagatgattc aggtgttagt ttaatgaata aaaatgatga tgatattgat 1080gactataatt taaggtggtt tttatttcct attgatgata atcaatatat tattacaagc 1140tacgcagcga ataattgtaa ggtttggaat gttaataatg ataaaataaa tgtttcaact 1200tattcttcaa caaactcgat acagaaatgg caaataaaag ctaatgcttc ttcgtatgta 1260atacaaagta ataatgggaa agttctaaca gcaggaaccg gtcaatctct tggattaata 1320cgtttaacgg atgaatcacc agataatccc aatcaacaat ggaatttaac tcctgtacaa 1380acaattcaac tcccaccaaa acctacaata gatacaaagt taaaagatta ccccaaatat 1440tcacaaactg gcaatataga caagggaaca cctcctcaat taatgggatg gacattaata 1500ccttgtatta tggtaaatga tccaaatata gataaaaaca ctcaaatcaa aactactcca 1560tattatattt taaaaaaata tcaatattgg caacaagcag taggaagtaa tgtagcttta 1620cgtccgcatg aaaaaaaatc atatgcttat gagtggggta cagaaataga tcaaaaaaca 1680actatcatta atacattagg atttcagatt aatatagatt cgggaatgaa atttgatata 1740ccagaagtag gtggaggtac agatgaaata aaaacacaat taaacgaaga attaaaaata 1800gaatatagcc gtgaaaccaa aataatggaa aaatatcagg aacaatcaga gatagataat 1860ccaactgatc aatcaatgaa ttctatagga ttcctcacta ttacttcttt agaattatat 1920cgatataatg gttcggaaat tagtgtaatg aaaattcaaa cttcagataa tgatacttac 1980aatgtgacct cttatccaga tcatcaacaa gctctattac ttcttacaaa tcattcatat 2040gaagaagtag aagaaataac aaatattccc aaaatatcac tgaaaaaatt aaaaaaatat 2100tatttttaaa acataattat attttgatag ctttttaaaa ataaagattg ttcaaagtaa 2160aatgaaagaa aatcttttat gaaactttaa tacaataaaa gaggaatatt ttcttataag 2220tacttccttg 2230 35 372 DNA Bacillus thuringiensis 35 atgtcagcacgtgaagtaca cattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180 atatattatagtataaatgg agaagcagaa attagtttat attttgacaa tccttattca 240 ggttctaataaatatgatgg gcattccaat aaaaatcaat atgaagttat tacccaagga 300 ggatcaggaaatcaatctca tgttacgtat actattcaaa ctgtatcttc acgatatggg 360 aataattcat aa372 36 123 PRT Bacillus thuringiensis 36 Met Ser Ala Arg Glu Val His IleAsp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp LysThr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val AlaAsn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met ThrGly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu IleSer Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr AspGly His Ser Asn Lys Asn Gln Tyr Glu Val 85 90 95 Ile Thr Gln Gly Gly SerGly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Val Ser SerArg Tyr Gly Asn Asn Ser 115 120 37 1152 DNA Bacillus thuringiensis 37acttatttaa gtttagatga ttcaggtgtt agtttaatga ataaaaatga tgatgatatt 120gatgactata atttaaggtg gtttttattt cctattgatg ataatcaata tattattaca 180acttattctt caacaaactc gatacagaaa tggcaaataa aagctaatgc ttcttcgtat 300gtaatacaaa gtaataatgg gaaagttcta acagcaggaa ccggtcaatc tcttggatta 360caaacaattc aactcccacc aaaacctaca atagatacaa agttaaaaga ttaccccaaa 480tattcacaaa ctggcaatat agacaaggga acacctcctc aattaatggg atggacatta 540ccatattata ttttaaaaaa atatcaatat tggcaacaag cagtaggaag taatgtagct 660ttacgtccgc atgaaaaaaa atcatatgct tatgagtggg gtacagaaat agatcaaaaa 720ataccagaag taggtggagg tacagatgaa ataaaaacac aattaaacga agaattaaaa 840atagaatata gccgtgaaac caaaataatg gaaaaatatc aggaacaatc agagatagat 900tatcgatata atggttcgga aattagtgta atgaaaattc aaacttcaga taatgatact 1020tacaatgtga cctcttatcc agatcatcaa caagctctat tacttcttac aaatcattca 1080tattattttt aa 1152 38 383 PRT Bacillus thuringiensis 38 Met Leu Asp ThrAsn Lys Ile Tyr Glu Ile Ser Asn Tyr Ala Asn Gly 1 5 10 15 Leu His AlaAla Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn LysAsn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Arg Trp Phe 35 40 45 Leu Phe ProIle Asp Asp Asn Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn CysLys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr TyrSer Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Ala SerSer Tyr Val Ile Gln Ser Asn Asn Gly Lys Val Leu Thr Ala 100 105 110 GlyThr Gly Gln Ser Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Pro 115 120 125Asp Asn Pro Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln 130 135140 Leu Pro Pro Lys Pro Thr Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145150 155 160 Tyr Ser Gln Thr Gly Asn Ile Asp Lys Gly Thr Pro Pro Gln LeuMet 165 170 175 Gly Trp Thr Leu Ile Pro Cys Ile Met Val Asn Asp Pro AsnIle Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile LeuLys Lys Tyr 195 200 205 Gln Tyr Trp Gln Gln Ala Val Gly Ser Asn Val AlaLeu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Ala Tyr Glu Trp Gly ThrGlu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly PheGln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu ValGly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu LeuLys Ile Glu Tyr Ser Arg Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr GlnGlu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn SerIle Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg TyrAsn Gly Ser Glu Ile Ser Val Met Lys Ile Gln Thr Ser 325 330 335 Asp AsnAsp Thr Tyr Asn Val Thr Ser Tyr Pro Asp His Gln Gln Ala 340 345 350 LeuLeu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365Asn Ile Pro Lys Ile Ser Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 38039 2132 DNA Bacillus thuringiensis 39 gtatttcagg gggtgaagat tcaagtaagtttattgatgt atattatcct tttgaagata 60 gtaattttaa atattatcaa tttataaaagtagcaattga ttttgatatt aatgaagatt 120 ttattaattt taataatcat gacaatatagggatatttaa ttttgttaca cgaaattttt 180 tattaaataa tgaaaatgat gaataaaaaatttaatttgt ttattatgtt tattttttga 240 aaattgaatg catatattaa tcgagtatgtataataaatt ttaattttat ggaggttgat 300 atttatgtca gcacgtgaag tacacattgatgtaaataat aagacaggtc atacattaca 360 attagaagat aaaacaaaac ttgatggtggtagatggcga acatcaccta caaatgttgc 420 taatgatcaa attaaaacat ttgtagcagaatcaaatggt tttatgacag gtacagaagg 480 tactatatat tatagtataa atggagaagcagaaattagt ttatattttg acaatccttt 540 tgcaggttct aataaatatg atggacattccaataaatct caatatgaaa ttattaccca 600 aggaggatca ggaaatcaat ctcatgttacgtatactatt caaaccacat cctcacgata 660 tgggcataaa tcataacaaa taattttttacgaaaatacc aaaaaataaa tattttttgg 720 tattttctaa tataaattac aaatatattaataataaaat tataagaaaa ggtgataaag 780 attatgttag atactaataa agtttatgaaataagcaatc atgctaatgg actatatgca 840 gcaacttatt taagtttaga tgattcaggtgttagtttaa tgaataaaaa tgatgatgat 900 attgatgatt ataacttaaa atggtttttatttcctattg atgatgatca atatattatt 960 acaagctatg cagcaaataa ttgtaaagtttggaatgtta ataatgataa aataaatgtt 1020 tcgacttatt cttcaacaaa ttcaatacaaaaatggcaaa taaaagctaa tggttcttca 1080 tatgtaatac aaagtgataa tggaaaagtcttaacagcag gaaccggtca agctcttgga 1140 ttgatacgtt taactgatga atcctcaaataatcccaatc aacaatggaa tttaacttct 1200 gtacaaacaa ttcaacttcc acaaaaacctataatagata caaaattaaa agattatccc 1260 aaatattcac caactggaaa tatagataatggaacatctc ctcaattaat gggatggaca 1320 ttagtacctt gtattatggt aaatgatccaaatatagata aaaatactca aattaaaact 1380 actccatatt atattttaaa aaaatatcaatattggcaac gagcagtagg aagtaatgta 1440 gctttacgtc cacatgaaaa aaaatcatatacttatgaat ggggcacaga aatagatcaa 1500 aaaacaacaa ttataaatac attaggatttcaaatcaata tagattcagg aatgaaattt 1560 gatataccag aagtaggtgg aggtacagatgaaataaaaa cacaactaaa tgaagaatta 1620 aaaatagaat atagtcatga aactaaaataatggaaaaat atcaagaaca atctgaaata 1680 gataatccaa ctgatcaatc aatgaattctataggatttc ttactattac ttccttagaa 1740 ttatatagat ataatggctc agaaattcgtataatgcaaa ttcaaacctc agataatgat 1800 acttataatg ttacttctta tccaaatcatcaacaagctt tattacttct tacaaatcat 1860 tcatatgaag aagtagaaga aataacaaatattcctaaaa gtacactaaa aaaattaaaa 1920 aaatattatt tttaaatatt gaaattagaaattatctaaa acaaaacgaa agataattta 1980 atctttaatt atttgtaaga taatcgtattttatttgtat taatttttat acaatataaa 2040 gtaatatctg tacgtgaaat tggtttcgcttcaatatcta atctcatctc atgtattaca 2100 tgcgtaatac cttcttgttc tgcttctacaag 2132 40 372 DNA Bacillus thuringiensis 40 atgtcagcac gtgaagtacacattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaa caaaacttgatggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaatta aaacatttgtagcagaatca aatggtttta tgacaggtac agaaggtact 180 atatattata gtataaatggagaagcagaa attagtttat attttgacaa tccttttgca 240 ggttctaata aatatgatggacattccaat aaatctcaat atgaaattat tacccaagga 300 ggatcaggaa atcaatctcatgttacgtat actattcaaa ccacatcctc acgatatggg 360 cataaatcat aa 372 41 123PRT Bacillus thuringiensis 41 Met Ser Ala Arg Glu Val His Ile Asp ValAsn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr LysLeu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn AspGln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser Asn Gly Phe Met Thr Gly ThrGlu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser LeuTyr Phe Asp Asn Pro Phe Ala 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly HisSer Asn Lys Ser Gln Tyr Glu Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly AsnGln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg TyrGly His Lys Ser 115 120 42 1241 DNA Bacillus thuringiensis misc_feature(18)..(18) Any nucleotide 42 wcdmtkdvrm wahkcmdndb ygtrawbmkg cwtkctgyhdcywagmawtd cvnwmhasrt 60 nchhtmsnwr manrgarcrr nwrgarhatg ttagatactaataaagttta tgaaataagc 120 aatcatgcta atggactata tgcagcaact tatttaagtttagatgattc aggtgttagt 180 ttaatgaata aaaatgatga tgatattgat gattataacttaaaatggtt tttatttcct 240 attgatgatg atcaatatat tattacaagc tatgcagcaaataattgtaa agtttggaat 300 gttaataatg ataaaataaa tgtttcgact tattcttcaacaaattcaat acaaaaatgg 360 caaataaaag ctaatggttc ttcatatgta atacaaagtgataatggaaa agtcttaaca 420 gcaggaaccg gtcaagctct tggattgata cgtttaactgatgaatcctc aaataatccc 480 aatcaacaat ggaatttaac ttctgtacaa acaattcaacttccacaaaa acctataata 540 gatacaaaat taaaagatta tcccaaatat tcaccaactggaaatataga taatggaaca 600 tctcctcaat taatgggatg gacattagta ccttgtattatggtaaatga tccaaatata 660 gataaaaata ctcaaattaa aactactcca tattatattttaaaaaaata tcaatattgg 720 caacgagcag taggaagtaa tgtagcttta cgtccacatgaaaaaaaatc atatacttat 780 gaatggggca cagaaataga tcaaaaaaca acaattataaatacattagg atttcaaatc 840 aatatagatt caggaatgaa atttgatata ccagaagtaggtggaggtac agatgaaata 900 aaaacacaac taaatgaaga attaaaaata gaatatagtcatgaaactaa aataatggaa 960 aaatatcaag aacaatctga aatagataat ccaactgatcaatcaatgaa ttctatagga 1020 tttcttacta ttacttcctt agaattatat agatataatggctcagaaat tcgtataatg 1080 caaattcaaa cctcagataa tgatacttat aatgttacttcttatccaaa tcatcaacaa 1140 gctttattac ttcttacaaa tcattcatat gaagaagtagaagaaataac aaatattcct 1200 aaaagtacac taaaaaaatt aaaaaaatat tatttttaav v1241 43 383 PRT Bacillus thuringiensis 43 Met Leu Asp Thr Asn Lys ValTyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr TyrLeu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp AspAsp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp AspAsp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val TrpAsn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser ThrAsn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr ValIle Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly GlnAla Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn ProAsn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu ProGln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg ProHis 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile AspGln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile AsnIle Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly GlyThr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile GluTyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln SerGlu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly PheLeu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly SerGlu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr TyrAsn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu LeuThr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile ProLys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 44 360 DNAArtificial Sequence Maize-optimized gene sequence encoding theapproximately 14 kDa toxin of 80JJ1 44 atgtccgccc gcgaggtgca catcgagatcaacaacaaga cccgccacac cctccagctc 60 gaggacaaga ccaagctctc cggcggcaggtggcgcacct ccccgaccaa cgtggcccgc 120 gacaccatca agacgttcgt ggcggagtcccacggcttca tgaccggcgt cgagggcatc 180 atctacttct ccgtgaacgg cgacgccgagatctccctcc acttcgacaa cccgtacatc 240 ggctccaaca agtgcgacgg ctcctccgacaagcccgagt acgaggtgat cacccagtcc 300 ggctccggcg acaagtccca cgtgacctacaccatccaga ccgtgtccct ccgcctctga 360 45 1158 DNA Artificial SequenceMaize-optimized gene sequence encoding the approximately 44 kDa toxin of80JJ1 45 atgctcgaca ccaacaaggt gtacgagatc tccaacctcg ccaacggcctctacacctcc 60 acctacctct ccctcgacga ctccggcgtg tccctcatgt ccaagaaggacgaggacatc 120 gacgactaca acctcaagtg gttcctcttc ccgatcgaca acaaccagtacatcatcacc 180 tcctacggcg ccaacaactg caaggtgtgg aacgtgaaga acgacaagatcaacgtgtcc 240 acctactcct ccaccaactc cgtgcagaag tggcagatca aggccaaggactcctcctac 300 atcatccagt ccgacaacgg caaggtgctc accgcgggcg tgggccagtccctcggcatc 360 gtgcgcctca ccgacgagtt cccggagaac tccaaccagc aatggaacctcaccccggtg 420 cagaccatcc agctcccgca gaagccgaag atcgacgaga agctcaaggaccacccggag 480 tactccgaga ccggcaacat caacccgaag accaccccgc agctcatgggctggaccctc 540 gtgccgtgca tcatggtgaa cgactccaag atcgacaaga acacccagatcaagaccacc 600 ccgtactaca tcttcaagaa atacaagtac tggaacctcg ccaagggctccaacgtgtcc 660 ctcctcccgc accagaagcg cagctacgac tacgagtggg gcaccgagaagaaccagaag 720 accaccatca tcaacaccgt gggcctgcag atcaacatcg actcggggatgaagttcgag 780 gtgccggagg tgggcggcgg caccgaggac atcaagaccc agctcaccgaggagctgaag 840 gtggagtact ccaccgagac caagatcatg accaagtacc aggagcactccgagatcgac 900 aacccgacca accagccgat gaactccatc ggcctcctca tctacacctccctcgagctg 960 taccgctaca acggcaccga gatcaagatc atggacatcg agacctccgaccacgacacc 1020 tacaccctca cctcctaccc gaaccacaag gaggcgctgc tgctgctgaccaaccactcc 1080 tacgaggagg tggaggagat caccaagatc ccgaagcaca ccctcatcaagctcaagaag 1140 cactacttca agaagtga 1158 46 24 DNA Artificial SequenceDNA sequence of a reverse primer 46 gtagaagcag aacaagaagg tatt 24 47 25DNA Artificial Sequence DNA sequence of a forward primer 47 atgtcagcwcgygaagtwca yattg 25 48 23 DNA Artificial Sequence DNA sequence of areverse primer 48 gtytgaathg tatahgthac atg 23 49 25 DNA ArtificialSequence DNA sequence of a forward primer 49 atgttagata cwaataaart wtatg25 50 29 DNA Artificial Sequence DNA sequence of a reverse primer 50gtwatttctt cwacttcttc atahgaatg 29 51 341 DNA Artificial Sequence DNAsequence from PS131W2 which encodes the 14 kDa protein 51 atgtcaggtcgagaagtaca tattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180 atatattttagtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaataaatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggagataaatctca tgtaacatat actattcaga c 341 52 113 PRT Bacillus thuringiensisMISC_FEATURE (389)..(389) Undetermined in the deduced amino acidsequence 52 Met Ser Gly Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr ArgHis 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly ArgTrp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr PheVal Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile TyrPhe Ser 50 55 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn ProTyr Ile 65 70 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro GluTyr Glu Val 85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val ThrTyr Thr Ile 100 105 110 Gln 53 1103 DNA Bacillus thuringiensis 53atgttagata caaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatcm 60acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120gatgattaca atttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatc tcttggaata 360gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720acamctatta ttaatacagt aggattgcaa attaatatag actcaggaat gaaatttgaa 780gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900aatccaacta atcaaccaat gaattctata ggacttctta tttacacttc tttagaatta 960tatcgatata acggtacaga aattaagata atggacatag aaacttcaga tcatgatact 1020tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattca 1080tatgaagaag tagaagaaat aac 1103 54 367 PRT Bacillus thuringiensisMISC_FEATURE (242)..(242) Undetermined in the deduced amino acidsequence 54 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala AsnGly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly ValSer Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn Leu LysTrp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser TyrGly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Ile AsnVal Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln IleLys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly Lys ValLeu Thr Ala 100 105 110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg Leu ThrAsp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr ProVal Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Glu LysLeu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile AsnPro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro CysIle Met Val Asn Asp Ser Lys Ile Asp 180 185 190 Lys Asn Thr Gln Ile LysThr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn LeuAla Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220 Gln Lys Arg SerTyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230 235 240 Thr XaaIle Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255 MetLys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265 270Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu Glu Leu305 310 315 320 Tyr Arg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp Ile GluThr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn HisLys Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu ValGlu Glu Ile 355 360 365 55 341 DNA Bacillus thuringiensis 55 atgtcagctcgtgaagtaca tattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatttgt agcagaatca catggtttta tgacaggtac agaaggtcat 180 atatattatagtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240 ggttctaataaatatgatgg ggattccaat aaacctcaat atgaagttac tacccaagga 300 ggatcaggaaatcaatctca tgtaacatat acgattcaaa c 341 56 113 PRT Bacillus thuringiensis56 Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly His Ile Tyr Tyr Ser 5055 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly Asp Ser Asn Lys Pro Gln Tyr Glu Val85 90 95 Thr Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile100 105 110 Gln 57 1103 DNA Bacillus thuringiensis misc_feature(1028)..(1028) Unknown 57 atgttagata ctaataaagt ttatgaaata agtaatcatgctaatggact atatgcagca 60 acttatttaa gtttagatga ttcaggtgtt agtttaatgaataaaaatga tgatgatatt 120 gatgattaca acttaaaatg gtttttattt cctattgatgatgatcaata tattattaca 180 agctatgcag caaataattg taaagtttgg aatgttaataatgataaaat aaatgtttcg 240 acttattctt taacaaattc aatacaaaaa tggcaaataaaagctaatgg ttcttcatat 300 gtaatacaaa gtgataatgg aaaagtctta acagcaggaaccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatc ttcaaataat cccaatcaacaatggaattt aacttctgta 420 caaacaattc aacttccaca aaaacctata atagatacaaaattaaaaga ttatcccaaa 480 tattcaccaa ctggaaatat agataatgga acatctcctcaattaatggg atggacatta 540 gtaccttgta ttatggtaaa tgatccaaat atagataaaaatactcaaat taaaactact 600 ccatattata ttttaaaaaa atatcaatat tggcaacgagcagtaggaag taatgtagct 660 ttacgtccac atgaaaaaaa atcatatact tatgaatggggaacagaaat agatcaaaaa 720 acaacaatca taaatacatt aggatttcaa atcaatatagattcaggaat gaaatttgat 780 ataccagaag taggtggagg tacagatgaa ataaaaacacaactaaatga agaattaaaa 840 atagaatata gtcgtgaaac taaaataatg gaaaaatatcaagaacaatc tgaaatagat 900 aatccaactg atcaaccaat gaattctata ggatttcttactattacttc tttagaatta 960 tatagatata atggctcaga aattcgtata atgcaaattcaaacctcaga taatgatact 1020 tataatgnta cttcttatcc agatcatcaa caagctttattacttcttac aaatcattca 1080 tatgaagaac tagaagaaat aac 1103 58 367 PRTBacillus thuringiensis MISC_FEATURE (343)..(343) Undetermined in thededuced amino acid sequence 58 Met Leu Asp Thr Asn Lys Val Tyr Glu IleSer Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser LeuAsp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile AspAsp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln TyrIle Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val AsnAsn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Leu Thr Asn Ser IleGln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln SerAsp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu GlyLeu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln GlnTrp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys ProIle Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser ProThr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly TrpThr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 LysAsn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp SerGly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp GluIle Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser ArgGlu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile AspAsn Pro Thr Asp 290 295 300 Gln Pro Met Asn Ser Ile Gly Phe Leu Thr IleThr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile ArgIle Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Xaa ThrSer Tyr Pro Asp His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn HisSer Tyr Glu Glu Leu Glu Glu Ile 355 360 365 59 340 DNA Bacillusthuringiensis 59 atgtcagcag gtgaagtaca tattgatgca aataataaga caggtcatacattacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaatgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtacagaaggtcat 180 atatattata gtataaatgg agaagcagaa attagtttat attttgataatccttattca 240 ggttctaata aatatgatgg ggattccaat aaacctcaat atgaagttactacccaagga 300 ggatcaggaa atcaatctca tgttacttat acaattcaaa 340 60 113PRT Bacillus thuringiensis 60 Met Ser Ala Gly Glu Val His Ile Asp AlaAsn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr LysLeu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn AspGln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly ThrGlu Gly His Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser LeuTyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly AspSer Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly AsnGln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 61 340 DNA Bacillusthuringiensis 61 tgtcagcacg tgaagtacat attgaaataa acaataaaac acgtcatacattacaattag 60 aggataaaac taaacttagc ggcggtagat ggcgaacatc acctacaaatgttgctcgtg 120 atacaattaa aacatttgta gcagaatcac atggttttat gacaggagtagaaggtatta 180 tatattttag tgtaaacgga gacgcagaaa ttagtttaca ttttgacaatccttatatag 240 gttctaataa atgtgatggt tcttctgata aacctgaata tgaagttattactcaaagcg 300 gatcaggaga taaatctcat gtgacatata cgattcagac 340 62 112PRT Bacillus thuringiensis 62 Ser Ala Arg Glu Val His Ile Glu Ile AsnAsn Lys Thr Arg His Thr 1 5 10 15 Leu Gln Leu Glu Asp Lys Thr Lys LeuSer Gly Gly Arg Trp Arg Thr 20 25 30 Ser Pro Thr Asn Val Ala Arg Asp ThrIle Lys Thr Phe Val Ala Glu 35 40 45 Ser His Gly Phe Met Thr Gly Val GluGly Ile Ile Tyr Phe Ser Val 50 55 60 Asn Gly Asp Ala Glu Ile Ser Leu HisPhe Asp Asn Pro Tyr Ile Gly 65 70 75 80 Ser Asn Lys Cys Asp Gly Ser SerAsp Lys Pro Glu Tyr Glu Val Ile 85 90 95 Thr Gln Ser Gly Ser Gly Asp LysSer His Val Thr Tyr Thr Ile Gln 100 105 110 63 1114 DNA Bacillusthuringiensis 63 atgttagata ctaataaaat ttatgaaata agcaatcttg ctaatggattatatacatca 60 acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaaggatgaagatatt 120 gatgattaca atttaaaatg gtttttattt cctattgata ataatcaatatattattaca 180 agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaataaatgtttca 240 acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaagattcttcatat 300 ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatctcttggaata 360 gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaatttaactcctgta 420 caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaagatcatcctgaa 480 tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatgggatggacatta 540 gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaattaaaactact 600 ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaagtaatgtatct 660 ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaaaaatcaaaaa 720 acaactatta ttaatacagt aggattgcaa attaatatag attcaggaatgaaatttgaa 780 gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactgaagaattaaaa 840 gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactcagagatagat 900 aatccaacta atcaaccaat gaattctata ggacttctta tttatacttctttagaatta 960 tatcgatata acggtacaga aattaagata atggacatag aaacttcagatcatgatact 1020 tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcacaaaccattct 1080 tatgaagaac tagaacaaat tacaagggcg aatt 1114 64 371 PRTBacillus thuringiensis 64 Met Leu Asp Thr Asn Lys Ile Tyr Glu Ile SerAsn Leu Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu AspAsp Ser Gly Val Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile Asp AspTyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr IleIle Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys AsnAsp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Val GlnLys Trp Gln Ile Lys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln Ser AspAsn Gly Lys Val Leu Thr Ala 100 105 110 Gly Val Gly Gln Ser Leu Gly IleVal Arg Leu Thr Asp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln Gln TrpAsn Leu Thr Pro Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro LysIle Asp Glu Lys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser Glu ThrGly Asn Ile Asn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly Trp ThrLeu Val Pro Cys Ile Met Val Asn Asp Ser Lys Ile Asp 180 185 190 Lys AsnThr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205 LysTyr Trp Asn Leu Ala Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220Gln Lys Arg Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230235 240 Thr Thr Ile Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp IleLys 260 265 270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr GluThr Lys 275 280 285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile Asp AsnPro Thr Asn 290 295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile Tyr ThrSer Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Thr Glu Ile Lys IleMet Asp Ile Glu Thr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr SerTyr Pro Asn His Lys Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn His SerTyr Glu Glu Leu Glu Gln Ile Thr 355 360 365 Arg Ala Asn 370 65 360 DNABacillus thuringiensis 65 atgtcagctc gcgaagtaca cattgaaata aacaataaaacacgtcatac attacaatta 60 gaggataaaa ctaaacttag cggcggtaga tggcgaacatcacctacaaa tgttgctcgt 120 gatacaatta aaacatttgt agcagaatca catggttttatgacaggagt agaaggtatt 180 atatatttta gtgtaaacgg agacgcagaa attagtttacattttgacaa tccttatata 240 ggttctaata aatgtgatgg ttcttctgat aaacctgaatatgaagttat tactcaaagc 300 ggatcaggag ataaatctca tgtgacatat actattcagacagtatcttt acgattataa 360 66 119 PRT Bacillus thuringiensis 66 Met SerAla Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15 ThrLeu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30 ThrSer Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45 GluSer His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 50 55 60 ValAsn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 65 70 75 80Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile 100 105110 Gln Thr Val Ser Leu Arg Leu 115 67 1158 DNA Bacillus thuringiensis67 atgttagata ctaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatca 60acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120gatgattaca atttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatc tcttggaata 360gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720acaactatta ttaatacagt aggattgcaa attaatatag attcaggaat gaaatttgaa 780gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900aatccaacta atcaaccaat gaattctata ggacttctta tttatacttc tttagaatta 960tatcgatata acggtacaga aattaagata atggacatag aaacttcaga tcatgatact 1020tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattcg 1080tatgaagaag tagaagaaat aacaaaaata cctaagcata cacttataaa attgaaaaaa 1140cattatttta aaaaataa 1158 68 385 PRT Bacillus thuringiensis 68 Met LeuAsp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala Asn Gly 1 5 10 15 LeuTyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 MetSer Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 LeuPhe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 AsnAsn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Ile Asn Val Ser 65 70 75 80Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Phe Pro 115120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Glu Lys Leu Lys Asp His ProGlu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Asn Pro Lys Thr Thr ProGln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn AspSer Lys Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr TyrIle Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn Leu Ala Lys Gly Ser AsnVal Ser Leu Leu Pro His 210 215 220 Gln Lys Arg Ser Tyr Asp Tyr Glu TrpGly Thr Glu Lys Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr ValGly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Glu Val ProGlu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265 270 Thr Gln Leu Thr GluGlu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280 285 Ile Met Thr LysTyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Pro MetAsn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu Glu Leu 305 310 315 320 TyrArg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp Ile Glu Thr Ser 325 330 335Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Lys Glu Ala 340 345350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355360 365 Lys Ile Pro Lys His Thr Leu Ile Lys Leu Lys Lys His Tyr Phe Lys370 375 380 Lys 385 69 341 DNA Bacillus thuringiensis 69 atgtcagcacgagaagtaca cattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatctgt agcagaatca aatggtttta tgacaggtac agaaggtact 180 atatattatagtataaatgg agaagcagaa attagtttat attttgacaa tccttttgca 240 ggttctaataaatatgatgg acattccaat aaatctcaat atgaaattat tacccaagga 300 ggatcaggaaatcaatctca tgttacttat acaattcaga c 341 70 113 PRT Bacillus thuringiensis70 Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Ser Val Ala 3540 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 5055 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile100 105 110 Gln 71 340 DNA Bacillus thuringiensis 71 atgtcagcaggcgaagttca tattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatttgt agcagaatca aatggtttta tgacaggtac agaaggtact 180 atatattatagtataaatgg agaagcagaa attagtttat attttgacaa tccttttgca 240 ggttctaataaatatgatgg acattccaat aaatctcaat atgaaattat tacccaagga 300 ggatcaggaaatcaatctca tgtaacgtat acaattcaaa 340 72 113 PRT Bacillus thuringiensis72 Met Ser Ala Gly Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 3540 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 5055 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile100 105 110 Gln 73 340 DNA Bacillus thuringiensis 73 atgtcagctcgcgaagtwca tattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180 atatattttagtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaataaatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggagataaatctca tgtgacatat accattcaaa 340 74 113 PRT Bacillus thuringiensis74 Met Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 5055 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 6570 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile100 105 110 Gln 75 341 DNA Bacillus thuringiensis 75 atgtcagctcgcgaagttca tattgaaata aataataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttac cagtggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggaat agaaggtatt 180 atatattttagcgtaaacgg agaagcagaa attagtttac attttgacaa tccttatgta 240 ggttctaataaatatgatgg ttcttctgat aaagctgcat acgaagttat tgctcaaggt 300 ggatcaggggatatatctca tgtaacttat acaattcaaa c 341 76 113 PRT Bacillus thuringiensis76 Met Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Thr Ser Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Ile Glu Gly Ile Ile Tyr Phe Ser 5055 60 Val Asn Gly Glu Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Val 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly Ser Ser Asp Lys Ala Ala Tyr Glu Val85 90 95 Ile Ala Gln Gly Gly Ser Gly Asp Ile Ser His Val Thr Tyr Thr Ile100 105 110 Gln 77 1175 DNA Bacillus thuringiensis 77 atgttagatactaataaagt ttatgaaata agcaatcatg ctaatggatt atatacatca 60 acttatttaagtctggatga ttcaggtgtt agtttaatgg gtcaaaatga tgaggatata 120 gatgaatmcaatttaaagtg gttcttattt ccaatagata ataatcaata tattattaca 180 agctatggagcgaataattg taaagtttgg aatgttaaaa atgataaagt aaatgtttca 240 acgtattctccaacaaactc agtacaaaaa tggcaaataa aagctaaaaa ttcttcatat 300 ataatacaaagtgagaatgg aaaagtctta acagcaggaa taggtcaatc tcctggaata 360 gtacgcttaaccgatgaatc atcagagagt tctaaccaac aatggaattt aatccctgta 420 caaacaatttcactcccaca aaaacctaaa atagataaaa aattaaaaga tcatcctgaa 480 tattcagaaaccggaaatat agctactgga acaattcctc aattaatggg atggacatta 540 gtaccttgtattatggtaaa tgatccaaaa atagataaaa acactcaaat taaaactact 600 ccatattatatttttaaaaa atatcaatac tggaaacgag caataggaag taatgtatct 660 ttacttccacatcaaaaaaa atcatatgat tatgagtggg gtacagaaga aaatcaaaaa 720 acaactattattaatacagt aggatttcaa attaatgtag attcaggaat gaagtttgag 780 gtaccagaagtaggaggagg tacagaagaa ataaaaacac aattaaatga agaattaaaa 840 gttgaatatagcactgacac caaaataatg aaaaaatatc aagaacactc agagatagat 900 aatccaactaatcaaacaat gaattctata ggatttctta cttttacttc tttagaatta 960 tatcgatataacggttcgga aattcgtata atgagaatgg aaacttcaga taatgatact 1020 tatactctgacctcttatcc aaatcataga gaagcattat tacttctcac aaatcattca 1080 tatcaagaagtacmagaaat tacaagggcg aattcttgca gatatccatc acactggcgg 1140 gccggtcgagccttgcatct agaggggccc caatt 1175 78 391 PRT Bacillus thuringiensisMISC_FEATURE (43)..(43) Undetermined in the deduced amino acid sequence78 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 510 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 2025 30 Met Gly Gln Asn Asp Glu Asp Ile Asp Glu Xaa Asn Leu Lys Trp Phe 3540 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 5055 60 Asn Asn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Val Asn Val Ser 6570 75 80 Thr Tyr Ser Pro Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys85 90 95 Asn Ser Ser Tyr Ile Ile Gln Ser Glu Asn Gly Lys Val Leu Thr Ala100 105 110 Gly Ile Gly Gln Ser Pro Gly Ile Val Arg Leu Thr Asp Glu SerSer 115 120 125 Glu Ser Ser Asn Gln Gln Trp Asn Leu Ile Pro Val Gln ThrIle Ser 130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Lys Lys Leu Lys AspHis Pro Glu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Ala Thr Gly ThrIle Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met ValAsn Asp Pro Lys Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr ProTyr Tyr Ile Phe Lys Lys Tyr 195 200 205 Gln Tyr Trp Lys Arg Ala Ile GlySer Asn Val Ser Leu Leu Pro His 210 215 220 Gln Lys Lys Ser Tyr Asp TyrGlu Trp Gly Thr Glu Glu Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile AsnThr Val Gly Phe Gln Ile Asn Val Asp Ser Gly 245 250 255 Met Lys Phe GluVal Pro Glu Val Gly Gly Gly Thr Glu Glu Ile Lys 260 265 270 Thr Gln LeuAsn Glu Glu Leu Lys Val Glu Tyr Ser Thr Asp Thr Lys 275 280 285 Ile MetLys Lys Tyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 GlnThr Met Asn Ser Ile Gly Phe Leu Thr Phe Thr Ser Leu Glu Leu 305 310 315320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Arg Met Glu Thr Ser 325330 335 Asp Asn Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Arg Glu Ala340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Gln Glu Val Xaa Glu IleThr 355 360 365 Arg Ala Asn Ser Cys Arg Tyr Pro Ser His Trp Arg Ala GlyArg Ala 370 375 380 Leu His Leu Glu Gly Pro Gln 385 390 79 341 DNABacillus thuringiensis 79 atgtcagcag gtgaagttca tattgaaata aataataaaacacgtcatac attacaatta 60 gaggataaaa ctaaacttac cagtggtaga tggcgaacatcacctacaaa tgttgctcgt 120 gatacaatta aaacatttgt agcagaatca catggttttatgacaggaat agaaggtatt 180 atatatttta gcgtaaacgg agaagcagaa attagtttacattttgacaa tccttatgta 240 ggttctaata aatatgatgg ttcttctgat aaagctgcatacgaagttat tgctcaaggt 300 ggatcagggg atatatctca tctaacatat acaattcaaa c341 80 113 PRT Bacillus thuringiensis 80 Met Ser Ala Gly Glu Val His IleGlu Ile Asn Asn Lys Thr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp LysThr Lys Leu Thr Ser Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val AlaArg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met ThrGly Ile Glu Gly Ile Ile Tyr Phe Ser 50 55 60 Val Asn Gly Glu Ala Glu IleSer Leu His Phe Asp Asn Pro Tyr Val 65 70 75 80 Gly Ser Asn Lys Tyr AspGly Ser Ser Asp Lys Ala Ala Tyr Glu Val 85 90 95 Ile Ala Gln Gly Gly SerGly Asp Ile Ser His Leu Thr Tyr Thr Ile 100 105 110 Gln 81 1410 DNABacillus thuringiensis 81 atgttagata ctaataaaat ttatgaaata agcaatcatgctaatggatt atatacatca 60 acttatttaa gtctggatga ttcaggtgtt agtttaatgggtcaaaatga tgaggatata 120 gatgaataca atttaaagtg gttcttattt ccaatagataataatcaata tattattaca 180 agctatggag cgaataattg taaagtttgg aatgttaaaaatgataaagt aaatgtttca 240 acgtattctc caacaaactc agtacaaaaa tggcaaataaaagctaaaaa ttcttcatat 300 ataatacaaa gtgagaatgg aaaagtctta acagcaggaataggtcaatc tcttggaata 360 gtacgcttaa ccgatgaatc atcagagagt tctaaccaacaatggaattt aatccctgta 420 caaacaattt cactcccaca aaaacctaaa atagataaaaaattaaaaga tcatcctgaa 480 tattcagaaa ccggaaatat agctactgga acaattcctcaattaatggg atggacatta 540 gtaccttgta ttatggtaaa tgatccaaaa ataggtaaaaacactcaaat taaaactact 600 ccatattata tttttaaaaa atatcaatac tggaaacgagcaataggaag taatgtatct 660 ttacttccac atcaaaaaaa atcatatgat tatgagtggggtacagaaga aaatcaaaaa 720 acaactatta ttaatacagt aggatttcaa attaatgtagattcaggaat gaagtttgag 780 gtaccagaag taggaggagg tacagaagaa ataaaaacacaattaaatga agaattaaaa 840 gttgaatata gcactgacac caaaataatg aaaaaatatcaagaacactc agagatagat 900 aatccaacta atcaaacaac gaattctata ggatttcttacttttacttc tttagaatta 960 tatcgatata acggttcgga aattcgtata atgagaatggaaacttcaga taatgatact 1020 tatactctga cctcttatcc aaatcataga gaagcattattacttctcac aaatcattct 1080 tatcaagaag taagccgaat tccagcacac tggcggccgttactagtgga tccgagctcg 1140 gtaccaagct tggcgtaatc atggtcatag stgtttcctgtgtgaaattg ttatccgctc 1200 acaattccac acaacatacg agccggaagc ataaagtgtaaagcctgggg tgcctaatga 1260 gtgagctaac tcacattaat tgcgttgcgc tcactgcccgctttccagtc gggaaacctg 1320 tcgtgccagc tgcattaatg aatcggccaa cgcgcggggagaggcggttt gcgtattggg 1380 cgctcttccg cttcctcgct cactgactcg 1410 82 462PRT Bacillus thuringiensis MISC_FEATURE (389)..(389) Undetermined in thededuced amino acid sequence 82 Met Leu Asp Thr Asn Lys Ile Tyr Glu IleSer Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser LeuAsp Asp Ser Gly Val Ser Leu 20 25 30 Met Gly Gln Asn Asp Glu Asp Ile AspGlu Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln TyrIle Ile Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val LysAsn Asp Lys Val Asn Val Ser 65 70 75 80 Thr Tyr Ser Pro Thr Asn Ser ValGln Lys Trp Gln Ile Lys Ala Lys 85 90 95 Asn Ser Ser Tyr Ile Ile Gln SerGlu Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Ile Gly Gln Ser Leu GlyIle Val Arg Leu Thr Asp Glu Ser Ser 115 120 125 Glu Ser Ser Asn Gln GlnTrp Asn Leu Ile Pro Val Gln Thr Ile Ser 130 135 140 Leu Pro Gln Lys ProLys Ile Asp Lys Lys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser GluThr Gly Asn Ile Ala Thr Gly Thr Ile Pro Gln Leu Met 165 170 175 Gly TrpThr Leu Val Pro Cys Ile Met Val Asn Asp Pro Lys Ile Gly 180 185 190 LysAsn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205Gln Tyr Trp Lys Arg Ala Ile Gly Ser Asn Val Ser Leu Leu Pro His 210 215220 Gln Lys Lys Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Glu Asn Gln Lys 225230 235 240 Thr Thr Ile Ile Asn Thr Val Gly Phe Gln Ile Asn Val Asp SerGly 245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu GluIle Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Val Glu Tyr Ser ThrAsp Thr Lys 275 280 285 Ile Met Lys Lys Tyr Gln Glu His Ser Glu Ile AspAsn Pro Thr Asn 290 295 300 Gln Thr Thr Asn Ser Ile Gly Phe Leu Thr PheThr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile ArgIle Met Arg Met Glu Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Thr Leu ThrSer Tyr Pro Asn His Arg Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn HisSer Tyr Gln Glu Val Ser Arg Ile Pro 355 360 365 Ala His Trp Arg Pro LeuLeu Val Asp Pro Ser Ser Val Pro Ser Leu 370 375 380 Ala Ser Trp Ser XaaPhe Pro Val Asn Cys Tyr Pro Leu Thr Ile Pro 385 390 395 400 His Asn IleArg Ala Gly Ser Ile Lys Cys Lys Ala Trp Gly Ala Val 405 410 415 Ser LeuThr Leu Ile Ala Leu Arg Ser Leu Pro Ala Phe Gln Ser Gly 420 425 430 AsnLeu Ser Cys Gln Leu His Ile Gly Gln Arg Ala Gly Arg Gly Gly 435 440 445Leu Arg Ile Gly Arg Ser Ser Ala Ser Ser Leu Thr Asp Ser 450 455 460 83340 DNA Bacillus thuringiensis 83 tgtcagcacg tgaagtacat attgatgtaaataataagac aggtcataca ttacaattag 60 aagataaaac aaaacttgat ggtggtagatggcgaacatc acctacaaat gttgctaatg 120 atcaaattaa aacatttgta gcagaatcaaatggttttat gacaggtaca gaaggtacta 180 tatattatag tataaatgga gaagcagaaattagtttata ttttgacaat ccttttgcag 240 gttctaataa atatgatgga cattccaataaatctcaata tgaaattatt acccaaggag 300 gatcaggaaa tcaatctcat gtgacatatactattcaaac 340 84 112 PRT Bacillus thuringiensis 84 Ser Ala Arg Glu ValHis Ile Asp Val Asn Asn Lys Thr Gly His Thr 1 5 10 15 Leu Gln Leu GluAsp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg Thr 20 25 30 Ser Pro Thr AsnVal Ala Asn Asp Gln Ile Lys Thr Phe Val Ala Glu 35 40 45 Ser Asn Gly PheMet Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser Ile 50 55 60 Asn Gly Glu AlaGlu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala Gly 65 70 75 80 Ser Asn LysTyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile Ile 85 90 95 Thr Gln GlyGly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile Gln 100 105 110 85 1114DNA Bacillus thuringiensis 85 atgttagata ctaataaagt ttatgaaataagcaatcatg ctaatggact atatgcagca 60 acttatttaa gtttagatga ttcaggtgttagtttaatga ataaaaatga tgatgatatt 120 gatgattata acttaaaatg gtttttatttcctattgatg atgatcaata tattattaca 180 agctatgcag caaataattg taaagtttggaatgttaata atgataaaat aaatgtttcg 240 acttattctt caacaaattc aatacaaaaatggcaaataa aagctaatgg ttcttcatat 300 gtaatacaaa gtgataatgg aaaagtcttaacagcaggaa ccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatc ctcaaataatcccaatcaac aatggaattt aacttctgta 420 caaacaattc aacttccacg aaaacctataatagatacaa aattaaaaga ttatcccaaa 480 tattcaccaa ctggaaatat agataatggaacatctcctc aattaatggg atggacatta 540 gtaccttgta ttatggtaaa tgatccaaatatagataaaa atactcaaat taaaactact 600 ccatattata ttttaaaaaa atatcaatattggcaacgag cagtaggaag taatgtagct 660 ttacgtccac atgaaaaaaa atcatatacttatgaatggg gcacagaaat agatcaaaaa 720 acaacaatta taaatacatt aggatttcaaatcaatatag attcaggaat gaaatttgat 780 ataccagaag taggtggagg tacagatgaaataaaaacac aactaaatga agaattaaaa 840 atagaatata gtcatgaaac taaaataatggaaaaatatc aagaacaatc tgaaatagat 900 aatccaactg atcaatcaat gaattctataggatttctta ctattacttc cttagaatta 960 tatagatata atggctcaga aattcgtataatgcaaattc aaacctcaga taatgatact 1020 tataatgtta cttcttatcc aaatcatcaacaagctttat tacttcttac aaatcattca 1080 tatgaagaag ttgaagaaat aacaagggcgaatt 1114 86 371 PRT Bacillus thuringiensis 86 Met Leu Asp Thr Asn LysVal Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala ThrTyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn AspAsp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile AspAsp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys ValTrp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser SerThr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser TyrVal Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr GlyGln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn AsnPro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 LeuPro Arg Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys LysTyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu ArgPro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu IleAsp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln IleAsn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly GlyGly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys IleGlu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu GlnSer Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile GlyPhe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn GlySer Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp ThrTyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu LeuLeu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Arg AlaAsn 370 87 341 DNA Bacillus thuringiensis 87 atgtcagctg gcgaagttcatattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaa ctaaacttagcggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaatta aaacatttgtagcagaatca catggtttta tgacaggagt agaaggtatt 180 atatatttta gtgtaaacggagacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaata aatgtgatggttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggag ataaatctcatgtcacttat acaattcaaa c 341 88 113 PRT Bacillus thuringiensis 88 Met SerAla Gly Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15 ThrLeu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30 ThrSer Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45 GluSer His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 50 55 60 ValAsn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 65 70 75 80Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile 100 105110 Gln 89 1186 DNA Bacillus thuringiensis 89 atgttagata caaataaagtttatgaaata agcaatcttg ctaatggatt atatacatca 60 acttatttaa gtcttgatgattcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120 gatgattaca atttaaaatggtttttattt cctattgata ataatcaata tattattaca 180 agctatggag ctaataattgtaaagtttgg aatgttaaaa atgataaaat aaatgtttca 240 acttattctt caacaaactctgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300 ataatacaaa gtgataatggaaaggtctta acagcaggag taggtcaatc tcttggaata 360 gtacgcctaa ctgatgaatttccagagaat tctaaccaac aatggaattt aactcctgta 420 caaacaattc aactcccacaaaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480 tattcagaaa ccggaaatataaatcctaaa acaactcctc aattaatggg atggacatta 540 gtaccttgta ttatggtaaatgattcaaaa atagataaaa acactcaaat taaaactact 600 ccatattata tttttaaaaaatataaatac tggaatctag caaaaggaag taatgtatct 660 ttacttccac atcaaaaaagatcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720 acaactatta ttaatacagtaggattgcaa attaatatag attcaggaat gaaatttgaa 780 gtaccagaag taggaggaggtacagaagac ataaaaacac aattaactga agaattaaaa 840 gttgaatata gcactgaaaccaaaataatg acgaaatatc aagaacactc agagatagat 900 aatccaacta atcaaccaatgaattctata ggacttctta tttatacttc tttagaatta 960 tatcgatata acggrcagaaattaagataa tggacataga aacttcagat catgatactt 1020 acactcttac ttcttatccaaatcataaag aagcattatt acttctcaca aaccattctt 1080 atgaagaagt agaagaaattacaagggcga attccagcac actggcggcc gttactagtg 1140 gatccgagct cggtaccaagcttggcgtgt caggtcaaag ggttca 1186 90 392 PRT Bacillus thuringiensis 90Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala Asn Gly 1 5 1015 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 2530 Met Ser Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 4045 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 5560 Asn Asn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Ile Asn Val Ser 65 7075 80 Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 8590 95 Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala100 105 110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu PhePro 115 120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr Pro Val Gln ThrIle Gln 130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Glu Lys Leu Lys AspHis Pro Glu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Asn Pro Lys ThrThr Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met ValAsn Asp Ser Lys Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr ProTyr Tyr Ile Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn Leu Ala Lys GlySer Asn Val Ser Leu Leu Pro His 210 215 220 Gln Lys Arg Ser Tyr Asp TyrGlu Trp Gly Thr Glu Lys Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile AsnThr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe GluVal Pro Glu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265 270 Thr Gln LeuThr Glu Glu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280 285 Ile MetThr Lys Tyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 GlnPro Met Asn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu Glu Leu 305 310 315320 Tyr Arg Tyr Asn Gly Gln Lys Leu Arg Trp Thr Lys Leu Gln Ile Met 325330 335 Ile Leu Thr Leu Leu Leu Leu Ile Gln Ile Ile Lys Lys His Tyr Tyr340 345 350 Phe Ser Gln Thr Ile Leu Met Lys Lys Lys Lys Leu Gln Gly ArgIle 355 360 365 Pro Ala His Trp Arg Pro Leu Leu Val Asp Pro Ser Ser ValPro Ser 370 375 380 Leu Ala Cys Gln Val Lys Gly Phe 385 390 91 341 DNABacillus thuringiensis 91 atgtcagcag ccgaagtaca tattgaaata ataaatcatacaggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattattacacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttttgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttacattttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgattataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaactt a341 92 113 PRT Bacillus thuringiensis 92 Met Ser Ala Ala Glu Val His IleGlu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys ArgThr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val ProAsn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu ThrGly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu IleThr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr SerGly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg AlaGlu His Arg Ala Asn Asn His Asp His Val 100 105 110 Thr 93 341 DNABacillus thuringiensis 93 atgtcagatc gcgaagtaca tattgaaata ataaatcatacaggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattattacacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttttgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttacattttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgattataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaactt a341 94 113 PRT Bacillus thuringiensis 94 Met Ser Asp Arg Glu Val His IleGlu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys ArgThr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val ProAsn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu ThrGly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu IleThr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr SerGly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg AlaGlu His Arg Ala Asn Asn His Asp His Val 100 105 110 Thr 95 353 DNABacillus thuringiensis 95 atgtcagcac gtgaagtaca tattgaaata ataaatcatacaggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattattacacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttttgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttacattttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgattataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaacatatacgattca aac 353 96 117 PRT Bacillus thuringiensis 96 Met Ser Ala ArgGlu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu GlnMet Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr ProVal Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser AspGly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn GlyGlu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly SerAsn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile ThrGlu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105 110 ThrTyr Thr Ile Gln 115 97 353 DNA Bacillus thuringiensis 97 atgtcagctcgtgaagtaca tattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaactagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctgatttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttatactataaatgg agaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaataaatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaacatagagctaa taatcatgat catgtgacat atacaattca aac 353 98 117 PRT Bacillusthuringiensis 98 Met Ser Ala Arg Glu Val His Ile Glu Ile Ile Asn His ThrGly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His GlyGlu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp LeuPhe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile IleIle Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe Asp AsnPro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp AspAsp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn AsnHis Asp His Val 100 105 110 Thr Tyr Thr Ile Gln 115 99 353 DNA Bacillusthuringiensis 99 atgtcaggtc gcgaagttca tattgaaata ataaatcata caggtcataccttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattatta cacccgtgaatgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttt tgacaggagtagaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttac attttgacaatccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttataactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaacat atacgattcaaac 353 100 117 PRT Bacillus thuringiensis 100 Met Ser Gly Arg Glu ValHis Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met AspLys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val AsnVal Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly ValLeu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu IleGlu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn LysTyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu AlaArg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105 110 Thr Tyr ThrIle Gln 115 101 353 DNA Bacillus thuringiensis 101 atgtcagctc gtgaagtacatattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgcacatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttcaagcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatggagaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaata aatattctggacgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaataatcatgat catgttacgt atacaattca aac 353 102 117 PRT Bacillusthuringiensis 102 Met Ser Ala Arg Glu Val His Ile Glu Ile Ile Asn HisThr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala HisGly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser AspLeu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly IleIle Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe AspAsn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser AspAsp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu His Arg Ala AsnAsn His Asp His Val 100 105 110 Thr Tyr Thr Ile Gln 115 103 353 DNABacillus thuringiensis 103 atgtcaggtc gcgaagtaga tattgaaata ataaatcatacaggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattattacacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttttgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttacattttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgattataaagttat aactgaagcg 300 agagcagaac atagagctaa taatcatgat catgtaacatatactattca gac 353 104 117 PRT Bacillus thuringiensis 104 Met Ser GlyArg Glu Val Asp Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr LeuGln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile ThrPro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly SerAsp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile AsnGly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 GlySer Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 IleThr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105 110Thr Tyr Thr Ile Gln 115 105 353 DNA Bacillus thuringiensis 105atgtcagcac gtgaagtaca tattgaaata ataaatcata caggtcatac cttacaaatg 60gataaaagaa ctagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120aattcttctg atttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180ataatttata ctataaatgg agaaatagaa attaccttac attttgacaa tccttatgca 240ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300agagcagaac atagagctaa taatcatgat catgtaacat ataccattca aac 353 106 117PRT Bacillus thuringiensis 106 Met Ser Ala Arg Glu Val His Ile Glu IleIle Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr ArgLeu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn AsnSer Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly ValGlu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr LeuHis Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly ArgSer Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu HisArg Ala Asn Asn His Asp His Val 100 105 110 Thr Tyr Thr Ile Gln 115 107341 DNA Bacillus thuringiensis 107 atgtcaggtc gcgaagttca tattgatgtaaataataaga caggtcatac attacaatta 60 gaagataaaa caagacttga tggtggtagatggcgaacat cacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatcacatggtttta tgacaggtac agaaggtact 180 atatattata gtataaatgg agaagcagaaattagtttat attttgacaa tccttattca 240 ggttctaata aatatgatgg gcattccaataaaaatcaat atgaagttat tacccaagga 300 ggatcaggaa atcaatctca tctgacgtatacaattcaaa c 341 108 113 PRT Bacillus thuringiensis 108 Met Ser Gly ArgGlu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu GlnLeu Glu Asp Lys Thr Arg Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser ProThr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser HisGly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn GlyGlu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly SerAsn Lys Tyr Asp Gly His Ser Asn Lys Asn Gln Tyr Glu Val 85 90 95 Ile ThrGln Gly Gly Ser Gly Asn Gln Ser His Leu Thr Tyr Thr Ile 100 105 110 Gln109 1114 DNA Bacillus thuringiensis 109 atgttagata ctaataaagt atatgaaataagtaattatg ctaatggatt acatgcagca 60 acttatttaa gtttagatga ttcaggtgttagtttaatga ataaaaatga tgatgatatt 120 gatgactata atttaaggtg gtttttatttcctattgatg ataatcaata tattattaca 180 agctacgcag cgaataattg taaggtttggaatgttaata atgataaaat aaatgtttca 240 acttattctt caacaaactc gatacagaaatggcaaataa aagctaatgc ttcttcgtat 300 gtaatacaaa gtaataatgg gaaagttctaacagcaggaa ccggtcaatc tcttggatta 360 atacgtttaa cggatgaatc accagataatcccaatcaac aatggaattt aactcctgta 420 caaacaattc aactcccacc aaaacctacaatagatacaa agttaaaaga ttaccccaaa 480 tattcacaaa ctggcaatat agacaagggaacacctcctc aattaatggg atggacatta 540 ataccttgta ttatggtaaa tgatccaaatatagataaaa acactcaaat caaaactact 600 ccatattata ttttaaaaaa atatcaatattggcaacaag cagtaggaag taatgtagct 660 ttacgtccgc atgaaaaaaa atcatatgcttatgagtggg gtacagaaat agatcaaaaa 720 acaactatca ttaatacatt aggatttcagattaatatag attcgggaat ggaatttgat 780 ataccagaag taggtggagg tacagatgaaataaaaacac aattaaacga agaattaaaa 840 atagaatata gccgtgaaac caaaataatggaaaaatatc aggaacaatc agagatagat 900 aatccaactg atcaatcaat gaattctataggattcctca ctattacttc tttagaatta 960 tatcgatata atggttcgga aattagtgtaatgaaaattc aaacttcaga taatgatact 1020 tacaatgtga cctcttatcc agatcatcaacaagctctat tacttcttac aaatcattca 1080 tatgaacaag tacaagaaat aacaagggcgaatt 1114 110 371 PRT Bacillus thuringiensis 110 Met Leu Asp Thr Asn LysVal Tyr Glu Ile Ser Asn Tyr Ala Asn Gly 1 5 10 15 Leu His Ala Ala ThrTyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn AspAsp Asp Ile Asp Asp Tyr Asn Leu Arg Trp Phe 35 40 45 Leu Phe Pro Ile AspAsp Asn Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys ValTrp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser SerThr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Ala Ser Ser TyrVal Ile Gln Ser Asn Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr GlyGln Ser Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Pro 115 120 125 Asp AsnPro Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln 130 135 140 LeuPro Pro Lys Pro Thr Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155160 Tyr Ser Gln Thr Gly Asn Ile Asp Lys Gly Thr Pro Pro Gln Leu Met 165170 175 Gly Trp Thr Leu Ile Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys LysTyr 195 200 205 Gln Tyr Trp Gln Gln Ala Val Gly Ser Asn Val Ala Leu ArgPro His 210 215 220 Glu Lys Lys Ser Tyr Ala Tyr Glu Trp Gly Thr Glu IleAsp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln IleAsn Ile Asp Ser Gly 245 250 255 Met Glu Phe Asp Ile Pro Glu Val Gly GlyGly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys IleGlu Tyr Ser Arg Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu GlnSer Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile GlyPhe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn GlySer Glu Ile Ser Val Met Lys Ile Gln Thr Ser 325 330 335 Asp Asn Asp ThrTyr Asn Val Thr Ser Tyr Pro Asp His Gln Gln Ala 340 345 350 Leu Leu LeuLeu Thr Asn His Ser Tyr Glu Gln Val Gln Glu Ile Thr 355 360 365 Arg AlaAsn 370 111 341 DNA Bacillus thuringiensis 111 atgtcagctc gtgaagtacatattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaa ctaaacttagcggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaatta aaacatttgtagcagaatca catggtttta tgacaggagt agaaggtatt 180 atatatttta gtgtaaacggagacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaata aatgtgatggttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggag ataaatctcatgttacatat acaattcaga c 341 112 113 PRT Bacillus thuringiensis 112 MetSer Ala Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 50 55 60Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 65 70 7580 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 9095 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile 100105 110 Gln 113 360 DNA Bacillus thuringiensis 113 atgtcagctc gcgaagtacacattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaa ctaaacttagcggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaatta aaacatttgtagcagaatca catggtttta tgacaggagt agaaggtatt 180 atatatttta gtgtaaacggagacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaata aatgtgatggttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggag ataaatctcatgtgacatat actattcaga cagtatcttt acgattataa 360 114 119 PRT Bacillusthuringiensis 114 Met Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn LysThr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser GlyGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly IleIle Tyr Phe Ser 50 55 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe AspAsn Pro Tyr Ile 65 70 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp LysPro Glu Tyr Glu Val 85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser HisVal Thr Tyr Thr Ile 100 105 110 Gln Thr Val Ser Leu Arg Leu 115 115 1158DNA Bacillus thuringiensis 115 atgttagata ctaataaagt ttatgaaataagcaatcttg ctaatggatt atatacatca 60 acttatttaa gtcttgatga ttcaggtgttagtttaatga gtaaaaagga tgaagatatt 120 gatgattaca atttaaaatg gtttttatttcctattgata ataatcaata tattattaca 180 agctatggag ctaataattg taaagtttggaatgttaaaa atgataaaat aaatgtttca 240 acttattctt caacaaactc tgtacaaaaatggcaaataa aagctaaaga ttcttcatat 300 ataatacaaa gtgataatgg aaaggtcttaacagcaggag taggtgaatc tcttggaata 360 gtacgcctaa ctgatgaatt tccagagaattctaaccaac aatggaattt aactcctgta 420 caaacaattc aactcccaca aaaacctaaaatagatgaaa aattaaaaga tcatcctgaa 480 tattcagaaa ccggaaatat aaatcctaaaacaactcctc aattaatggg atggacatta 540 gtaccttgta ttatggtaaa tgattcaggaatagataaaa acactcaaat taaaactact 600 ccatattata tttttaaaaa atataaatactggaatctag caaaaggaag taatgtatct 660 ttacttccac atcaaaaaag atcatatgattatgaatggg gtacagaaaa aaatcaaaaa 720 acatctatta ttaatacagt aggattgcaaattaatatag attcaggaat gaaatttgaa 780 gtaccagaag taggaggagg tacagaagacataaaaacac aattaactga agaattaaaa 840 gttgaatata gcactgaaac caaaataatgacgaaatatc aagaacactc agagatagat 900 aatccaacta atcaaccaat gaattctataggacttctta tttatacttc tttagaatta 960 tatcgatata acggtacaga aattaagataatggacatag aaacttcaga tcatgatact 1020 tacactctta cttcttatcc aaatcataaagaagcattat tacttctcac aaaccattcg 1080 tatgaagaag tagaagaaat aacaaaaatacctaagcata cacttataaa attgaaaaaa 1140 cattatttta aaaaataa 1158 116 385PRT Bacillus thuringiensis 116 Met Leu Asp Thr Asn Lys Val Tyr Glu IleSer Asn Leu Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser LeuAsp Asp Ser Gly Val Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile AspAsp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln TyrIle Ile Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val LysAsn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser ValGln Lys Trp Gln Ile Lys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln SerAsp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Val Gly Glu Ser Leu GlyIle Val Arg Leu Thr Asp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln GlnTrp Asn Leu Thr Pro Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys ProLys Ile Asp Glu Lys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser GluThr Gly Asn Ile Asn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly TrpThr Leu Val Pro Cys Ile Met Val Asn Asp Ser Gly Ile Asp 180 185 190 LysAsn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205Lys Tyr Trp Asn Leu Ala Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215220 Gln Lys Arg Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225230 235 240 Thr Ser Ile Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp SerGly 245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu AspIle Lys 260 265 270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser ThrGlu Thr Lys 275 280 285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile AspAsn Pro Thr Asn 290 295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile TyrThr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Thr Glu Ile LysIle Met Asp Ile Glu Thr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu ThrSer Tyr Pro Asn His Lys Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn HisSer Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Lys Ile Pro Lys His ThrLeu Ile Lys Leu Lys Lys His Tyr Phe Lys 370 375 380 Lys 385 117 341 DNABacillus thuringiensis 117 atgtcagcac gccaacttca tattgatgta aataataagacaggtcatac attacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacatcacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggttttatgacaggtac agaaggtact 180 atatattata gtataaatgg agaagcagaa attagtttatattttgacaa tccttattca 240 ggttctaata aatatgatgg gcattctaat aaaaatcaatatgaagttat tacccaagga 300 ggatcaggaa atcaatctca tgtgacttat acgattcaca c341 118 113 PRT Bacillus thuringiensis 118 Met Ser Ala Arg Gln Leu HisIle Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu AspLys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn ValAla Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe MetThr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala GluIle Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys TyrAsp Gly His Ser Asn Lys Asn Gln Tyr Glu Val 85 90 95 Ile Thr Gln Gly GlySer Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 His 119 341 DNABacillus thuringiensis 119 atgtcaggtc gtgaagttca tattgatgta aataataagacaggtcatac attacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacatcacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggttttatgacaggtac agaaggtact 180 atatattata gtataaatgg agaagcagaa attagtttatattttgataa tccttattca 240 ggttctaata aatatgatgg gcattccaat aaacctcaatatgaagttac tacccaagga 300 ggatcaggaa atcaatctca tgtaacgtat actattcaaa c341 120 113 PRT Bacillus thuringiensis 120 Met Ser Gly Arg Glu Val HisIle Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu AspLys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn ValAla Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe MetThr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala GluIle Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys TyrAsp Gly His Ser Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly GlySer Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 121 341 DNABacillus thuringiensis 121 atgtcaggtc gcgaagttga cattgatgta aataataagacaggtcatac attacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacatcacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggttttatgacaggtac agaaggtact 180 atatattata gtataaatgg agaagcagaa attagtttatattttgataa tccttattca 240 ggttctaata aatatgatgg gcattccaat aaacctcaatatgaagttac tacccaagga 300 ggatcaggaa atcaatctca tgtcacatat acgattcaaa c341 122 113 PRT Bacillus thuringiensis 122 Met Ser Gly Arg Glu Val AspIle Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu AspLys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn ValAla Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe MetThr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala GluIle Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys TyrAsp Gly His Ser Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly GlySer Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 123 341 DNABacillus thuringiensis 123 atgtcagcac gtgaagtaga tattgatgta aataataagacaggtcatac attacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacatcacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggttttatgacaggtac agaaggtact 180 atatattata gtataaatgg agaagcagaa attagtttatattttgataa tccttattca 240 ggttctaata aatatgatgg gcattccaat aaacctcaatatgaagttac tacccaagga 300 ggatcaggaa atcaatctca tgtaacgtat acgattcaaa c341 124 113 PRT Bacillus thuringiensis 124 Met Ser Ala Arg Glu Val AspIle Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu AspLys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn ValAla Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe MetThr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala GluIle Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys TyrAsp Gly His Ser Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly GlySer Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 125 1103 DNABacillus thuringiensis 125 atgttagata ctaataaagt ttatgaaata agtaatcatgctaatggact atatgcagca 60 acttatttaa gtttagatga ttcaggtgtt agtttaatgaataaaaatga tgatgatatt 120 gatgattata acttaaaatg gtttttattt cctattgatgatgatcaata tattattaca 180 agctatgcag caaataattg taaagtttgg aatgttaataatgataaaat aaatgtttcg 240 acttattctt caacaaattc aatacaaaaa tggcaaataaaagctaatgg ttcttcatat 300 gtaatacaaa gtgataatgg aaaagtctta acagcaggaaccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatc ctcaaataat cccaatcaacaatggaattt aacttctgta 420 caaacaattc aacttccaca aaaacctata atagatacaaaattaaaaga ttatcccaaa 480 tattcaccaa ctggaaatat agataatgga acatctcctcaattaatggg atggacatta 540 gtaccttgta ttatggtaaa tgatccaaat atagataaaaatactcaaat taaaactact 600 ccatattata ttttaaaaaa atatcaatat tggcaacgagcagtaggaag taatgtagct 660 ttacgtccac atgaagaaaa atcatatact tatgaatggggaacagaaat agatcaaaaa 720 acaacaatca taaatacatt aggatttcaa atcaatatagattcaggaat gaaatttgat 780 ataccagaag taggtggagg tacagatgaa ataaaaacacaactaaatga agaattaaaa 840 atagaatata gtcgtgaaac taaaataatg gaaaaatatcaagaacaatc tgaaatagat 900 aatccaactg atcaaccaat gaattctata ggatttcttactattacttc tttagaatta 960 tatagatata atggctcaga aattcgtata atgcaaattcaaacctcaga taatgatact 1020 tataatgtta cttcttatcc agatcatcaa caagctttattacttcttac aaatcattca 1080 tatgaagaac ttgaagaaat tag 1103 126 367 PRTBacillus thuringiensis 126 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile SerAsn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu AspAsp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp AspTyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr IleIle Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn AsnAsp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile GlnLys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser AspAsn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly LeuIle Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln TrpAsn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro IleIle Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro ThrGly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp ThrLeu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys AsnThr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 GlnTyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220Glu Glu Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu IleLys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser Arg GluThr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp AsnPro Thr Asp 290 295 300 Gln Pro Met Asn Ser Ile Gly Phe Leu Thr Ile ThrSer Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg IleMet Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr SerTyr Pro Asp His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His SerTyr Glu Glu Leu Glu Glu Ile 355 360 365 127 369 DNA Artificial SequencePolynucleotide sequence for a gene designated 149B1-15-PO, which isoptimized for expression in Zea mays 127 atgtccgccc gcgaggtgcacatcgacgtg aacaacaaga ccggccacac cctccagctg 60 gaggacaaga ccaagctcgacggcggcagg tggcgcacct ccccgaccaa cgtggccaac 120 gaccagatca agaccttcgtggccgaatcc aacggcttca tgaccggcac cgagggcacc 180 atctactact ccatcaacggcgaggccgag atcagcctct acttcgacaa cccgttcgcc 240 ggctccaaca aatacgacggccactccaac aagtcccagt acgagatcat cacccagggc 300 ggctccggca accagtcccacgtgacctac accatccaga ccacctcctc ccgctacggc 360 cacaagtcc 369 128 1149DNA Artificial Sequence Polynucleotide sequence for a gene designated149B1-45-PO, which is optimized for expression in Zea mays 128atgctcgaca ccaacaaggt gtacgagatc agcaaccacg ccaacggcct ctacgccgcc 60acctacctct ccctcgacga ctccggcgtg tccctcatga acaagaacga cgacgacatc 120gacgactaca acctcaagtg gttcctcttc ccgatcgacg acgaccagta catcatcacc 180tcctacgccg ccaacaactg caaggtgtgg aacgtgaaca acgacaagat caacgtgtcc 240acctactcct ccaccaactc catccagaag tggcagatca aggccaacgg ctcctcctac 300gtgatccagt ccgacaacgg caaggtgctc accgccggca ccggccaggc cctcggcctc 360atccgcctca ccgacgagtc ctccaacaac ccgaaccagc aatggaacct gacgtccgtg 420cagaccatcc agctcccgca gaagccgatc atcgacacca agctcaagga ctacccgaag 480tactccccga ccggcaacat cgacaacggc acctccccgc agctcatggg ctggaccctc 540gtgccgtgca tcatggtgaa cgacccgaac atcgacaaga acacccagat caagaccacc 600ccgtactaca tcctcaagaa gtaccagtac tggcagaggg ccgtgggctc caacgtcgcg 660ctccgcccgc acgagaagaa gtcctacacc tacgagtggg gcaccgagat cgaccagaag 720accaccatca tcaacaccct cggcttccag atcaacatcg acagcggcat gaagttcgac 780atcccggagg tgggcggcgg taccgacgag atcaagaccc agctcaacga ggagctcaag 840atcgagtact cccacgagac gaagatcatg gagaagtacc aggagcagtc cgagatcgac 900aacccgaccg accagtccat gaactccatc ggcttcctca ccatcacctc cctggagctc 960taccgctaca acggctccga gatccgcatc atgcagatcc agacctccga caacgacacc 1020tacaacgtga cctcctaccc gaaccaccag caggccctgc tgctgctgac caaccactcc 1080tacgaggagg tggaggagat caccaacatc ccgaagtcca ccctcaagaa gctcaagaag 1140tactacttc 1149 129 357 DNA Artificial Sequence Polynucleotide sequencefor a gene designated 80JJ1-15-PO7, which is optimized for expression inmaize 129 atgtccgccc gcgaggtgca catcgagatc aacaacaaga cccgccacaccctccagctc 60 gaggacaaga ccaagctctc cggcggcagg tggcgcacct ccccgaccaacgtggcccgc 120 gacaccatca agacgttcgt ggcggagtcc cacggcttca tgaccggcgtcgagggcatc 180 atctacttct ccgtgaacgg cgacgccgag atctccctcc acttcgacaacccgtacatc 240 ggctccaaca agtccgacgg ctcctccgac aagcccgagt acgaggtgatcacccagtcc 300 ggctccggcg acaagtccca cgtgacctac accatccaga ccgtgtccctccgcctc 357 130 119 PRT Artificial Sequence Amino acid sequence for atoxin encoded by the gene designated 80JJ1-15-PO7 130 Met Ser Ala ArgGlu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15 Thr Leu GlnLeu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30 Thr Ser ProThr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser HisGly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 50 55 60 Val Asn GlyAsp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 65 70 75 80 Gly SerAsn Lys Ser Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95 Ile ThrGln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile 100 105 110 GlnThr Val Ser Leu Arg Leu 115 131 21 DNA Artificial SequenceOligonucleotide primer (15kfor1) 131 atgtcagctc gcgaagtaca c 21 132 22DNA Artificial Sequence Oligonucleotide primer (45krev6) 132 gtccatcccattaattgagg ag 22 133 399 DNA Bacillus thuringiensis 133 atgtcagcacgtgaagtaca cattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaactagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctgatttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttatactataaatgg agaaatagaa attcccttac attttgacaa tccttatgca 240 ggttctaataaatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaacatagagctaa taatcatgat catgtaacat atacagttca aagaaacata 360 tcacgatataccaataaatt atgttctaat aactcctaa 399 134 132 PRT Bacillus thuringiensis134 Met Ser Ala Arg Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 510 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 2025 30 Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 3540 45 Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 5055 60 Ile Asn Gly Glu Ile Glu Ile Pro Leu His Phe Asp Asn Pro Tyr Ala 6570 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val85 90 95 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val100 105 110 Thr Tyr Thr Val Gln Arg Asn Ile Ser Arg Tyr Thr Asn Lys LeuCys 115 120 125 Ser Asn Asn Ser 130 135 1164 DNA Bacillus thuringiensis135 atgatagaaa ctaataagat atatgaaata agcaataaag ctaatggatt atatgcaact 60acttatttaa gttttgataa ttcaggtgtt agtttattaa ataaaaatga atctgatatt 120aatgattata atttgaaatg gtttttattt cctattgata ataatcagta tattattaca 180agttatggag taaataaaaa taaggtttgg actgctaatg gtaataaaat aaatgttaca 240acatattccg cagaaaattc agcacaacaa tggcaaataa gaaacagttc ttctggatat 300ataatagaaa ataataatgg gaaaatttta acggcaggaa caggccaatc attaggttta 360ttatatttaa ctgatgaaat acctgaagat tctaatcaac aatggaattt aacttcaata 420caaacaattt cacttccttc acaaccaata attgatacaa cattagtaga ttaccctaaa 480tattcaacga ccggtagtat aaattataat ggtacagcac ttcaattaat gggatggaca 540ctcataccat gtattatggt atacgataaa acgatagctt ctacacacac tcaaattaca 600acaacccctt attatatttt gaaaaaatat caacgttggg tacttgcaac aggaagtggt 660ctatctgtac ctgcacatgt caaatcaact ttcgaatacg aatggggaac agacacagat 720caaaaaacca gtgtaataaa tacattaggt tttcaaatta atacagatac aaaattaaaa 780gctactgtac cagaagtagg tggaggtaca acagatataa gaacacaaat cactgaagaa 840cttaaagtag aatatagtag tgaaaataaa gaaatgcgaa aatataaaca aagctttgac 900gtagacaact taaattatga tgaagcacta aatgctgtag gatttattgt tgaaacttca 960ttcgaattat atcgaatgaa tggaaatgtc cttataacaa gtataaaaac tacaaataaa 1020gacacctata atacagttac ttatccaaat cataaagaag ttttattact tcttacaaat 1080cattcttatg aagaagtaac agcactaact ggcatttcca aagaaagact tcaaaatctt 1140aaaaacaatt ggaaaaaaag ataa 1164 136 387 PRT Bacillus thuringiensis 136Met Ile Glu Thr Asn Lys Ile Tyr Glu Ile Ser Asn Lys Ala Asn Gly 1 5 1015 Leu Tyr Ala Thr Thr Tyr Leu Ser Phe Asp Asn Ser Gly Val Ser Leu 20 2530 Leu Asn Lys Asn Glu Ser Asp Ile Asn Asp Tyr Asn Leu Lys Trp Phe 35 4045 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Val 50 5560 Asn Lys Asn Lys Val Trp Thr Ala Asn Gly Asn Lys Ile Asn Val Thr 65 7075 80 Thr Tyr Ser Ala Glu Asn Ser Ala Gln Gln Trp Gln Ile Arg Asn Ser 8590 95 Ser Ser Gly Tyr Ile Ile Glu Asn Asn Asn Gly Lys Ile Leu Thr Ala100 105 110 Gly Thr Gly Gln Ser Leu Gly Leu Leu Tyr Leu Thr Asp Glu IlePro 115 120 125 Glu Asp Ser Asn Gln Gln Trp Asn Leu Thr Ser Ile Gln ThrIle Ser 130 135 140 Leu Pro Ser Gln Pro Ile Ile Asp Thr Thr Leu Val AspTyr Pro Lys 145 150 155 160 Tyr Ser Thr Thr Gly Ser Ile Asn Tyr Asn GlyThr Ala Leu Gln Leu 165 170 175 Met Gly Trp Thr Leu Ile Pro Cys Ile MetVal Tyr Asp Lys Thr Ile 180 185 190 Ala Ser Thr His Thr Gln Ile Thr ThrThr Pro Tyr Tyr Ile Leu Lys 195 200 205 Lys Tyr Gln Arg Trp Val Leu AlaThr Gly Ser Gly Leu Ser Val Pro 210 215 220 Ala His Val Lys Ser Thr PheGlu Tyr Glu Trp Gly Thr Asp Thr Asp 225 230 235 240 Gln Lys Thr Ser ValIle Asn Thr Leu Gly Phe Gln Ile Asn Thr Asp 245 250 255 Thr Lys Leu LysAla Thr Val Pro Glu Val Gly Gly Gly Thr Thr Asp 260 265 270 Ile Arg ThrGln Ile Thr Glu Glu Leu Lys Val Glu Tyr Ser Ser Glu 275 280 285 Asn LysGlu Met Arg Lys Tyr Lys Gln Ser Phe Asp Val Asp Asn Leu 290 295 300 AsnTyr Asp Glu Ala Leu Asn Ala Val Gly Phe Ile Val Glu Thr Ser 305 310 315320 Phe Glu Leu Tyr Arg Met Asn Gly Asn Val Leu Ile Thr Ser Ile Lys 325330 335 Thr Thr Asn Lys Asp Thr Tyr Asn Thr Val Thr Tyr Pro Asn His Lys340 345 350 Glu Val Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val ThrAla 355 360 365 Leu Thr Gly Ile Ser Lys Glu Arg Leu Gln Asn Leu Lys AsnAsn Trp 370 375 380 Lys Lys Arg 385 137 341 DNA Bacillus thuringiensis137 atgtcagcag gtgaagttca tattgaaata aataataaaa cacgtcatac attacaatta 60gaggataaaa ctaaacttac cagtggtaga tggcgaacat cacctacaaa tgttgctcgt 120gatacaatta aaacatttgt agcagaatca catggtttta tgacaggaat agaaggtatt 180atatatttta gcgtaaacgg agaagcagaa attagtttac attttgacaa tccttatgta 240ggttctaata aatatgatgg ttcttctgat aaagctgcat acgaagttat tgctcaaggt 300ggatcagggg atatatctca tctaacatat acaattcaaa c 341 138 113 PRT Bacillusthuringiensis 138 Met Ser Ala Gly Glu Val His Ile Glu Ile Asn Asn LysThr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Thr SerGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Ile Glu Gly IleIle Tyr Phe Ser 50 55 60 Val Asn Gly Glu Ala Glu Ile Ser Leu His Phe AspAsn Pro Tyr Val 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly Ser Ser Asp LysAla Ala Tyr Glu Val 85 90 95 Ile Ala Gln Gly Gly Ser Gly Asp Ile Ser HisLeu Thr Tyr Thr Ile 100 105 110 Gln 139 1158 DNA Bacillus thuringiensis139 atgttagata ctaataaaat ttatgaaata agcaatcatg ctaatggatt atatacatca 60acttatttaa gtctggatga ttcaggtgtt agtttaatgg gtcaaaatga tgaggatata 120gatgaataca atttaaagtg gttcttattt ccaatagata ataatcaata tattattaca 180agctatggag cgaataattg taaagtttgg aatgttaaaa atgataaagt aaatgtttca 240acgtattctc caacaaactc agtacaaaaa tggcaaataa aagctaaaaa ttcttcatat 300ataatacaaa gtgagaatgg aaaagtctta acagcaggaa taggtcaatc tcttggaata 360gtacgcttaa ccgatgaatc atcagagagt tctaaccaac aatggaattt aatccctgta 420caaacaattt cactcccaca aaaacctaaa atagataaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat agctactgga acaattcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgatccaaaa ataggtaaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atatcaatac tggaaacgag caataggaag taatgtatct 660ttacttccac atcaaaaaaa atcatatgat tatgagtggg gtacagaaga aaatcaaaaa 720acaactatta ttaatacagt aggatttcaa attaatgtag attcaggaat gaagtttgag 780gtaccagaag taggaggagg tacagaagaa ataaaaacac aattaaatga agaattaaaa 840gttgaatata gcactgacac caaaataatg aaaaaatatc aagaacactc agagatagat 900aatccaacta atcaaacaac gaattctata ggatttctta cttttacttc tttagaatta 960tatcgatata acggttcgga aattcgtata atgagaatgg aaacttcaga taatgatact 1020tatactctga cctcttatcc aaatcataga gaagcattat tacttctcac aaatcattct 1080tatcaagaag taagccgaat tccagcacac tggcggccgt tactagtgga tccgagctcg 1140gtaccaagct tggcgtaa 1158 140 385 PRT Bacillus thuringiensis 140 Met LeuAsp Thr Asn Lys Ile Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 LeuTyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 MetGly Gln Asn Asp Glu Asp Ile Asp Glu Tyr Asn Leu Lys Trp Phe 35 40 45 LeuPhe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 AsnAsn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Val Asn Val Ser 65 70 75 80Thr Tyr Ser Pro Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95Asn Ser Ser Tyr Ile Ile Gln Ser Glu Asn Gly Lys Val Leu Thr Ala 100 105110 Gly Ile Gly Gln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Ser Ser 115120 125 Glu Ser Ser Asn Gln Gln Trp Asn Leu Ile Pro Val Gln Thr Ile Ser130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Lys Lys Leu Lys Asp His ProGlu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Ala Thr Gly Thr Ile ProGln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn AspPro Lys Ile Gly 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr TyrIle Phe Lys Lys Tyr 195 200 205 Gln Tyr Trp Lys Arg Ala Ile Gly Ser AsnVal Ser Leu Leu Pro His 210 215 220 Gln Lys Lys Ser Tyr Asp Tyr Glu TrpGly Thr Glu Glu Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr ValGly Phe Gln Ile Asn Val Asp Ser Gly 245 250 255 Met Lys Phe Glu Val ProGlu Val Gly Gly Gly Thr Glu Glu Ile Lys 260 265 270 Thr Gln Leu Asn GluGlu Leu Lys Val Glu Tyr Ser Thr Asp Thr Lys 275 280 285 Ile Met Lys LysTyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Thr ThrAsn Ser Ile Gly Phe Leu Thr Phe Thr Ser Leu Glu Leu 305 310 315 320 TyrArg Tyr Asn Gly Ser Glu Ile Arg Ile Met Arg Met Glu Thr Ser 325 330 335Asp Asn Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Arg Glu Ala 340 345350 Leu Leu Leu Leu Thr Asn His Ser Tyr Gln Glu Val Ser Arg Ile Pro 355360 365 Ala His Trp Arg Pro Leu Leu Val Asp Pro Ser Ser Val Pro Ser Leu370 375 380 Ala 385 141 399 DNA Bacillus thuringiensis 141 atgtcagatcgcgaagtaca tattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaactagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctgatttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttatactataaatgg agaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaataaatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaacatagagctaa taatcatgat catgtaacat atacagttca aagaaacata 360 tcacgatataccaataaatt atgttctaat aactcctaa 399 142 132 PRT Bacillus thuringiensis142 Met Ser Asp Arg Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 510 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 2025 30 Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 3540 45 Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 5055 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 6570 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val85 90 95 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val100 105 110 Thr Tyr Thr Val Gln Arg Asn Ile Ser Arg Tyr Thr Asn Lys LeuCys 115 120 125 Ser Asn Asn Ser 130 143 871 DNA Bacillus thuringiensis143 atgatagaaa ctaataagat atatgaaata agcaataaag ctaatggatt atatgcaact 60acttatttaa gttttgataa ttcaggtgtt agtttattaa ataaaaatga atctgatatt 120aatgattata atttgaaatg gtttttattt cctattgata ataatcagta tattattaca 180agttatggag taaataaaaa taaggtttgg actgctaatg gtaataaaat aaatgttaca 240acatattccg cagaaaattc agcacaacaa tggcaaataa gaaacagttc ttctggatat 300ataatagaaa ataataatgg gaaaatttta acggcaggaa caggccaatc attaggttta 360ttatatttaa ctgatgaaat acctgaagat tctaatcaac aatggaattt aacttcaata 420caaacaattt cacttccttc acaaccaata attgatacaa cattagtaga ttaccctaaa 480tattcaacga ccggtagtat aaattataat ggtacagcac ttcaattaat gggatggaca 540ctcataccat gtattatggt atacgataaa acgatagctt ctacacacac tcaaattaca 600acaacccctt attatatttt gaaaaaatat caacgttggg tacttgcaac aggaagtggt 660ctatctgtac ctgcacatgt caaatcaact ttcgaatacg aatggggaac agacacagat 720caaaaaacca gtgtaataaa tacattaggt tttcaaatta atacagatac aaaattaaaa 780gctactgtac cagaagtagg tggaggtaca acagatataa gaacacaaat cactgaagaa 840cttaaagtag aatatagtag tgaaaataaa g 871 144 290 PRT Bacillusthuringiensis 144 Met Ile Glu Thr Asn Lys Ile Tyr Glu Ile Ser Asn LysAla Asn Gly 1 5 10 15 Leu Tyr Ala Thr Thr Tyr Leu Ser Phe Asp Asn SerGly Val Ser Leu 20 25 30 Leu Asn Lys Asn Glu Ser Asp Ile Asn Asp Tyr AsnLeu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile ThrSer Tyr Gly Val 50 55 60 Asn Lys Asn Lys Val Trp Thr Ala Asn Gly Asn LysIle Asn Val Thr 65 70 75 80 Thr Tyr Ser Ala Glu Asn Ser Ala Gln Gln TrpGln Ile Arg Asn Ser 85 90 95 Ser Ser Gly Tyr Ile Ile Glu Asn Asn Asn GlyLys Ile Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ser Leu Gly Leu Leu TyrLeu Thr Asp Glu Ile Pro 115 120 125 Glu Asp Ser Asn Gln Gln Trp Asn LeuThr Ser Ile Gln Thr Ile Ser 130 135 140 Leu Pro Ser Gln Pro Ile Ile AspThr Thr Leu Val Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Thr Thr Gly SerIle Asn Tyr Asn Gly Thr Ala Leu Gln Leu 165 170 175 Met Gly Trp Thr LeuIle Pro Cys Ile Met Val Tyr Asp Lys Thr Ile 180 185 190 Ala Ser Thr HisThr Gln Ile Thr Thr Thr Pro Tyr Tyr Ile Leu Lys 195 200 205 Lys Tyr GlnArg Trp Val Leu Ala Thr Gly Ser Gly Leu Ser Val Pro 210 215 220 Ala HisVal Lys Ser Thr Phe Glu Tyr Glu Trp Gly Thr Asp Thr Asp 225 230 235 240Gln Lys Thr Ser Val Ile Asn Thr Leu Gly Phe Gln Ile Asn Thr Asp 245 250255 Thr Lys Leu Lys Ala Thr Val Pro Glu Val Gly Gly Gly Thr Thr Asp 260265 270 Ile Arg Thr Gln Ile Thr Glu Glu Leu Lys Val Glu Tyr Ser Ser Glu275 280 285 Asn Lys 290 145 372 DNA Bacillus thuringiensis 145atgtcagcac gtgaagtaca cattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240ggttctaata aatatgatgg gcattccaat aaacctcaat atgaagttac tacccaagga 300ggatcaggaa atcaatctca tgttacgtat actattcaaa ctgcatcttc acgatatggg 360aataactcat aa 372 146 123 PRT Bacillus thuringiensis 146 Met Ser Ala ArgGlu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu GlnLeu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser ProThr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser HisGly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn GlyGlu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly SerAsn Lys Tyr Asp Gly His Ser Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr ThrGln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 GlnThr Ala Ser Ser Arg Tyr Gly Asn Asn Ser 115 120 147 1152 DNA Bacillusthuringiensis 147 atgttagata ctaataaagt ttatgaaata agtaatcatg ctaatggactatatgcagca 60 acttatttaa gtttagatga ttcaggtgtt agtttaatga ataaaaatgatgatgatatt 120 gatgattata acttaaaatg gtttttattt cctattgatg atgatcaatatattattaca 180 agctatgcag caaataattg taaagtttgg aatgttaata atgataaaataaatgtttcg 240 acttattctt caacaaattc aatacaaaaa tggcaaataa aagctaatggttcttcatat 300 gtaatacaaa gtgataatgg aaaagtctta acagcaggaa ccggtcaagctcttggattg 360 atacgtttaa ctgatgaatc ctcaaataat cccaatcaac aatggaatttaacttctgta 420 caaacaattc aacttccaca aaaacctata atagatacaa aattaaaagattatcccaaa 480 tattcaccaa ctggaaatat agataatgga acatctcctc aattaatgggatggacatta 540 gtaccttgta ttatggtaaa tgatccaaat atagataaaa atactcaaattaaaactact 600 ccatattata ttttaaaaaa atatcaatat tggcaacgag cagtaggaagtaatgtagct 660 ttacgtccac atgaaaaaaa atcatatact tatgaatggg gaacagaaatagatcaaaaa 720 acaacaatca taaatacatt aggatttcaa atcaatatag attcaggaatgaaatttgat 780 ataccagaag taggtggagg tacagatgaa ataaaaacac aactaaatgaagaattaaaa 840 atagaatata gtcgtgaaac taaaataatg gaaaaatatc aagaacaatctgaaatagat 900 aatccaactg atcaaccaat gaattctata ggatttctta ctattacttctttagaatta 960 tatagatata atggctcaga aattcgtata atgcaaattc aaacctcagataatgatact 1020 tataatgtta cttcttatcc agatcatcaa caagctttat tacttcttacaaatcattca 1080 tatgaagaag tagaagaaat aacaaatatt cctaaaagta cactaaaaaaattaaaaaaa 1140 tattattttt aa 1152 148 383 PRT Bacillus thuringiensis148 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 510 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 2025 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 3540 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 5055 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 6570 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu SerSer 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln ThrIle Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys AspTyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly ThrSer Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met ValAsn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr ProTyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val GlySer Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr TyrGlu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile AsnThr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe AspIle Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln LeuAsn Glu Glu Leu Lys Ile Glu Tyr Ser Arg Glu Thr Lys 275 280 285 Ile MetGlu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 GlnPro Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asp His Gln Gln Ala340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu IleThr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr TyrPhe 370 375 380 149 354 DNA Bacillus thuringiensis 149 atgtcagctcgcgaagttca tattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaactagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctgatttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttatactataaatgg agaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaataaatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaacatagagctaa taatcatgat catgtgacat atacaattca aaca 354 150 113 PRTBacillus thuringiensis 150 Met Ser Ala Arg Glu Val His Ile Glu Ile IleAsn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg LeuAla His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn Asn SerSer Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly Val GluGly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu HisPhe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg SerSer Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu His ArgAla Asn Asn His Asp His Val 100 105 110 Thr 151 353 DNA Bacillusthuringiensis 151 atgtcagctc gtgaagttca tattgaaata ataaatcata caggtcataccttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattatta cacccgtgaatgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttt tgacaggagtagaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttac attttgacaatccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttataactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaacat atacaattcaaac 353 152 113 PRT Bacillus thuringiensis 152 Met Ser Ala Arg Glu ValHis Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met AspLys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val AsnVal Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly ValLeu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu IleGlu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn LysTyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu AlaArg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105 110 Thr 153 353DNA Bacillus thuringiensis 153 atgtcagcac gcgaagtaga tattgaaataataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaatggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttctgatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaaattaccttac attttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagtgatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgatcatgtgactt atacaattca aac 353 154 113 PRT Bacillus thuringiensis 154 MetSer Ala Arg Glu Val Asp Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 7580 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 9095 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100105 110 Thr 155 37 DNA Artificial Sequence Oligonucleotide primer(F1new) 155 aaatattatt ttatgtcagc acgtgaagta cacattg 37 156 40 DNAArtificial Sequence Oligonucleotide primer (R1new) 156 tctctggtaccttattatga tttatgccca tatcgtgagg 40 157 45 DNA Artificial SequenceOligonucleotide primer (F2new) 157 agagaactag taaaaaggag ataaccatgttagatactaa taaag 45 158 46 DNA Artificial Sequence Oligonucleotideprimer (R2new) 158 cgtgctgaca taaaataata tttttttaat ttttttagtg tacttt 46159 506 PRT Artificial Sequence Approximately 58 kDa fusion protein 159Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 1015 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 2530 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 4045 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 5560 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 7075 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 8590 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu SerSer 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln ThrIle Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys AspTyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly ThrSer Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met ValAsn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr ProTyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val GlySer Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr TyrGlu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile AsnThr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe AspIle Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln LeuAsn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile MetGlu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 GlnSer Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu IleThr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr TyrPhe Met 370 375 380 Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys ThrGly His Thr 385 390 395 400 Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp GlyGly Arg Trp Arg Thr 405 410 415 Ser Pro Thr Asn Val Ala Asn Asp Gln IleLys Thr Phe Val Ala Glu 420 425 430 Ser Asn Gly Phe Met Thr Gly Thr GluGly Thr Ile Tyr Tyr Ser Ile 435 440 445 Asn Gly Glu Ala Glu Ile Ser LeuTyr Phe Asp Asn Pro Phe Ala Gly 450 455 460 Ser Asn Lys Tyr Asp Gly HisSer Asn Lys Ser Gln Tyr Glu Ile Ile 465 470 475 480 Thr Gln Gly Gly SerGly Asn Gln Ser His Val Thr Tyr Thr Ile Gln 485 490 495 Thr Thr Ser SerArg Tyr Gly His Lys Ser 500 505 160 1521 DNA Artificial Sequence Fusiongene encoding the protein of SEQ ID NO159 160 atgttagata ctaataaagtttatgaaata agcaatcatg ctaatggact atatgcagca 60 acttatttaa gtttagatgattcaggtgtt agtttaatga ataaaaatga tgatgatatt 120 gatgattata acttaaaatggtttttattt cctattgatg atgatcaata tattattaca 180 agctatgcag caaataattgtaaagtttgg aatgttaata atgataaaat aaatgtttcg 240 acttattctt caacaaattcaatacaaaaa tggcaaataa aagctaatgg ttcttcatat 300 gtaatacaaa gtgataatggaaaagtctta acagcaggaa ccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatcctcaaataat cccaatcaac aatggaattt aacttctgta 420 caaacaattc aacttccacaaaaacctata atagatacaa aattaaaaga ttatcccaaa 480 tattcaccaa ctggaaatatagataatgga acatctcctc aattaatggg atggacatta 540 gtaccttgta ttatggtaaatgatccaaat atagataaaa atactcaaat taaaactact 600 ccatattata ttttaaaaaaatatcaatat tggcaacgag cagtaggaag taatgtagct 660 ttacgtccac atgaaaaaaaatcatatact tatgaatggg gcacagaaat agatcaaaaa 720 acaacaatta taaatacattaggatttcaa atcaatatag attcaggaat gaaatttgat 780 ataccagaag taggtggaggtacagatgaa ataaaaacac aactaaatga agaattaaaa 840 atagaatata gtcatgaaactaaaataatg gaaaaatatc aagaacaatc tgaaatagat 900 aatccaactg atcaatcaatgaattctata ggatttctta ctattacttc cttagaatta 960 tatagatata atggctcagaaattcgtata atgcaaattc aaacctcaga taatgatact 1020 tataatgtta cttcttatccaaatcatcaa caagctttat tacttcttac aaatcattca 1080 tatgaagaag tagaagaaataacaaatatt cctaaaagta cactaaaaaa attaaaaaaa 1140 tattatttta tgtcagcacgtgaagtacac attgatgtaa ataataagac aggtcataca 1200 ttacaattag aagataaaacaaaacttgat ggtggtagat ggcgaacatc acctacaaat 1260 gttgctaatg atcaaattaaaacatttgta gcagaatcaa atggttttat gacaggtaca 1320 gaaggtacta tatattatagtataaatgga gaagcagaaa ttagtttata ttttgacaat 1380 ccttttgcag gttctaataaatatgatgga cattccaata aatctcaata tgaaattatt 1440 acccaaggag gatcaggaaatcaatctcat gttacgtata ctattcaaac cacatcctca 1500 cgatatgggc ataaatcata a1521 161 23 DNA Artificial Sequence Primer 45kD5′ 161 gatratratcaatatattat tac 23 162 20 DNA Artificial Sequence Primer 45kD3′rc 162caaggtarta atgtccatcc 20 163 24 DNA Artificial Sequence Primer 45kD5′01163 gatgatgrtm rakwwattat trca 24 164 24 DNA Artificial Sequence primer45kD5′02 164 gatgatgrtm ratatattat trca 24 165 23 DNA ArtificialSequence Primer 45kD3′03 165 ggawgkrcdy twdtmccwtg tat 23 166 23 DNAArtificial Sequence primer 45kD3′04 166 ggawgkacry tadtaccttg tat 23

1. A polynucleotide that encodes a protein having toxin activity againstan insect wherein said polynucleotide was obtained by DNA recombinationafter random fragmentation of a nucleic acid sequence that encodes anamino acid sequence selected from the group consisting of SEQ ID NO: 76,SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:136,SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ IDNO:146, SEQ ID NO:148, SEQ ID No:150, SEQ ID NO: 152, seq id no: 154,and seq id no:
 159. 2. An isolated protein having toxin activity againstan insect wherein said protein comprises at least ten contiguous aminoacids of an amino acid sequence selected from the group consisting ofSEQ ID NO:76, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:80,SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100,SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ IDNO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:134,SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ IDNO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQID NO:154, and SEQ ID NO:159.
 3. The protein of claim 2 wherein saidprotein has a molecular weight of approximately 45 kDa.
 4. The proteinof claim 2 wherein said protein has a molecular weight of approximately15 kDa.
 5. The protein according to claim 2 wherein said proteincomprises at least 100 contiguous amino acids of said amino acidsequence.
 6. An isolated protein having toxin activity against an insectwherein said protein comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:76, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,SEQ ID NO:152, SEQ ID NO:154, and SEQ ID NO:159.
 7. The polynucleotideof claim 1 wherein said nucleic acid sequence is selected from the groupconsisting of SEQ ID NO:75, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65,SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77,SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87,SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:11, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107,SEQ ID NO:109, SEQ ID NO:11, SEQ ID NO:113, SEQ ID NO:115, SEQ IDNO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137,SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ IDNO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:161,SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, and SEQ IDNO:166.
 8. A protein encoded by a polynucleotide of claim
 1. 9. Anisolated polynucleotide that encodes a protein of claim
 2. 10. Atransgenic plant cell comprising a polynucleotide of claim
 1. 11. Atransgenic plant cell comprising a polynucleotide that encodes a proteinof claim
 2. 12. A transgenic plant that produces a protein of claim 2.13. A transgenic plant that produces a protein of claim
 8. 14. The plantcell of claim 10 wherein said cell is in a seed.
 15. The plant cell ofclaim 11 wherein said cell is in a seed.
 16. A transgenic bacterial cellcomprising a polynucleotide of claim
 1. 17. A transgenic bacterial cellcomprising a polynucleotide that encodes a protein of claim
 2. 18. Amethod of inhibiting an insect wherein said method comprises contactingsaid insect with a protein of claim
 2. 19. A method of inhibiting aninsect wherein said method comprises contacting said insect with aprotein encoded by a polynucleotide of claim
 1. 20. A biologically pureculture of a Bacillus thuringiensis isolate selected from the groupconsisting of PS185GG, PS187G1, PS187Y2, PS201G, PS201HH2, PS242K10,KB54A1-6, KR589, PS185L12, PS185W3, PS187L14, PS186FF, PS131W2, PS158T3,PS158X10, PS185FF, PS187F3, PS201H2, PS201L3, PS203G2, PS204G4, PS204G4,PS204I11, PS204J7, PS236B6, PS246P42, KR1209, and KR1369.